Methods and compositions for consumables

ABSTRACT

Methods and compositions for the production of non-meat consumable products are described herein. A meat substitute is described which is constructed from a muscle analog, a fat analog, and a connective tissue analog.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/238,769 filed on Jan. 3, 2019, which is a continuation of U.S.application Ser. No. 15/786,776 filed on Oct. 18, 2017, which is acontinuation of U.S. application Ser. No. 14/796,970 filed Jul. 10,2015, which is a continuation of, and claims the benefit of priorityunder 35 U.S.C. § 120 to, PCT/US2014/011361 filed Jan. 13, 2014, whichis a continuation of, and claims the benefit of priority under 35 U.S.C.§ 120 to, U.S. application Ser. No. 13/941,211, filed Jul. 12, 2013,which claims the benefit of priority under 35 U.S.C. § 119(e) to U.S.Application Ser. No. 61/908,634, filed Nov. 25, 2013, and to U.S.Application Ser. No. 61/751,816, filed Jan. 11, 2013; and thisapplication is related to the following co-pending patent applications:Application Serial No. PCT/US2012/46560; Application Serial NoPCT/US2012/46552; Application Ser. No. 61/876,676, filed Sep. 11, 2013;Application Ser. No. 61/751,818, filed Jan. 11, 2013, and ApplicationSer. No. 61/611,999, filed Mar. 16, 2012, all of which are incorporatedherein by reference.

TECHNICAL FIELD

This invention relates to consumable products and more particularly, tonon-animal based replicas of animal-based food products, that can beproduced, in some embodiments, by breaking down non-animal materialsinto their constituent parts and reassembling those parts into theconsumables.

BACKGROUND

Animal farming has a profound negative environmental impact. Currentlyit is estimated that 30% of Earth's land surface is dedicated to animalfarming and that livestock account for 20% of total terrestrial animalbiomass. Due to this massive scale, animal farming accounts for morethan 18% of net greenhouse gas emissions. Animal farming may be thelargest human source of water pollution, and animal farming is by farthe world's largest threat to biodiversity. It has been estimated thatif the world's human population could shift from a meat containing dietto a diet free of animal products, 26% of Earth's land surface would befreed for other uses. Furthermore the shift to a vegetarian diet wouldmassively reduce water and energy consumption.

The consumption of meat has a profound negative impact on human health.The health benefits of a vegetarian diet are well established. If thehuman population would shift to a more vegetarian diet, there would be adecrease in health care costs.

Hunger is a worldwide problem, yet the world's 4 major commodity crops(soybeans, maize, wheat, and rice) already supply more than 100% of thehuman population's requirements for calories and protein, includingevery essential amino acid.

Plant based meat substitutes have largely failed to cause a shift to avegetarian diet. The current state of the art for meat substitutecompositions involves the extrusion of soy/grain mixture, resulting inproducts which largely fail to replicate the experience of cooking andeating meat. Common limitations of these products are a texture andmouthfeel that are more homogenous than that of equivalent meatproducts. Furthermore, as the products must largely be sold pre-cooked,with artificial flavors and aromas built in, they fail to replicatearomas, flavors, and other key features associated with cooking meat. Asa result, these products appeal mainly to a limited consumer base thatis already committed to vegetarianism/veganism, but have failed toappeal to the larger consumer segment accustomed to eating meat.

Food is any substance that is either eaten or drunk by any animal,including humans, for nutrition or pleasure. It is usually of plant oranimal origin, and contains essential nutrients, such as carbohydrates,fats, proteins, vitamins, or minerals. The substance is ingested by anorganism and assimilated by the organism's cells in an effort to produceenergy, maintain life, or stimulate growth.

Food typically has its origin in a photosynthetic organism, typicallyfrom plants. Some food is obtained directly from plants; but evenanimals that are used as food sources are raised by feeding them foodderived from plants. Edible fungi and bacteria are used to transformmaterials from plants or animals into other food products, mushrooms,bread, yogurt and the like.

In most cases, the plant or animal is fractionated into a variety ofdifferent portions, depending upon the purpose of the food. Often,certain portions of the plant, such as the seeds or fruits, are morehighly prized by humans than others and these are selected for humanconsumption whilst other less desirable portions, such as the stalks ofgrasses, are typically used for feeding animals.

Animals are typically butchered into smaller cuts of meat with specificflavor and handling properties before consumption.

While many foods can be eaten raw, many also undergo some form ofpreparation for reasons of safety, palatability, texture, or flavor. Atthe simplest level, this may involve washing, cutting, trimming, oradding other foods or ingredients. It may also involve mixing, heatingor cooling or fermentation and individual foods may be combined withother food products to achieve the desired mix of properties.

In recent years, attempts have been made to bring scientific rigor tothe process of food preparation, under the fields of food science andmolecular gastronomy. Food science broadly studies the safety,microbiology, preservation, chemistry, engineering and physics of foodpreparation, whereas molecular gastronomy focuses on the use ofscientific tools such as liquid nitrogen, emulsifying agents such as soylecithin and gelling agents such as calcium alginates to transform foodproducts into unexpected forms.

However, the raw material is typically an entire organism (plant oranimal) or an isolated tissue such as a steak, the fruiting body of afungus, or the seed of a plant. In some cases, the isolated tissue ismodified before food preparation, such as making flour or isolating oilsand bulk proteins from seeds.

Despite that fact that all of these items comprise a mixture ofproteins, carbohydrates, fats, vitamins and minerals, the physicalarrangement of these materials in the original plant or animaldetermines the use to which the plant or animal tissue will be put.Disclosed herein are improved methods and composition for the productionof consumables.

SUMMARY

Provided herein are consumable products and methods of making the same.The consumables can be non-animal based consumable goods, e.g.,containing mainly plant or entirely-plant based proteins and/or fats,and can be in the form of a beverage (e.g., an alcoholic beverage suchas cream liquor, or a protein drink), a protein supplement, a baked good(e.g., a bread or a cookie), a condiment (e.g., a mayonnaise, amustard), a meat product, or a meat substitute product (e.g., a groundbeef product). For example, the protein drink can be a meal replacementbeverage, a beer supplemented with the protein, or a distilled alcoholicbeverage (e.g., vodka or rum) supplemented with the protein. Thecondiment can be mayonnaise. The meat product can be a pate, a sausage,or a meat substitute that can include a muscle replica, plant-basedadipose and/or connective tissue. Coacervates that include one or moreproteins can be used to help bind the ingredients to each other in theconsumable products (e.g., a ground beef product).

Accordingly, provided herein is a consumable product comprising anisolated and purified plant protein, wherein the isolated and purifiedplant protein has (i) a solubility in a solution of at least 25 g/L at atemperature between about 2° C. and about 32° C., wherein the solutionhas a pH between 3 and 8, and has a sodium chloride content of 0 to 300mM or (ii) a solubility in a solution of at least 1 mg/ml at atemperature of between 90° C. and 110° C., wherein the solution has a pHbetween 5 and 8 and has a sodium chloride content of 0 to 300 mM. Insome embodiments, the consumable product is a beverage, a proteinsupplement, a baked good, a condiment, a meat product, or a meatsubstitute product. In some embodiments, the beverage is an alcoholicbeverage or a protein drink. In some embodiments, the alcoholic beverageis a cream liquor. The cream liquor can further include a non-dairylipid emulsion, where the cream liquor is free of animal products. Insome embodiments, the protein drink is a meal replacement beverage, abeer supplemented with said protein, or a distilled alcoholic beveragesupplemented with the protein. A condiment can be a mayonnaise replica.In some embodiments, the meat product can be a pate, a sausage replica,or a meat substitute. In some embodiments, the isolated and purifiedplant protein is at least 10 kDa in size. In some embodiments, theisolated and purified plant protein is not fully denatured. In somecases, the isolated and purified plant protein is not derived from soy.In some embodiments, the isolated and purified plant protein comprisesone or more of RuBisCo, moong 8S globulin, a pea globulin, a peaalbumin, a lentil protein, zein, or an oleosin.

In some embodiments, the isolated and purified plant protein comprises adehydrin, a hydrophilin, an intrinsically disordered protein, or aprotein identified based on its ability to stay soluble after boiling ata pH and a salt concentration comparable to a food. In some embodiments,the consumable product further comprises a plant derived lipid or amicrobial-derived lipid. In some embodiments, the consumable productfurther includes a second isolated and purified protein, and/or aseasoning agent, a flavoring agent, an emulsifier, a gelling agent, asugar, or a fiber.

The disclosure also provides a consumable product comprising acoacervate comprising one or more isolated and purified proteins. Insome embodiments, the consumable product is a meat replica. In someembodiments, the consumable product further includes a plant derivedlipid or a microbial-derived lipid. The plant derived lipid ormicrobial-derived lipid can comprise lecithin and/or an oil. The productcan include up to about 1% lecithin by weight. The product can includelecithin and the oil. In some embodiments, the oil is canola oil, palmoil, or cocoa butter. The product can include about 1% to about 9% ofthe oil. The one or more isolated and purified proteins can compriseplant proteins. The one or more plant proteins can comprise one or morepea proteins, chickpea proteins, lentil proteins, lupine proteins, otherlegume proteins, or mixtures thereof. In some embodiments, the one ormore pea proteins are legumins, vicilin, a convicilin, or a mixturethereof.

The disclosure also provides a meat replica comprising a muscle replica,a connective tissue replica, an adipose tissue replica, and a coacervatecomprising one or more isolated and purified proteins. The coacervatefurther can comprise a plant-derived lipid or microbial-derived lipid.The plant derived lipid or microbial-derived lipid can be lecithinand/or an oil. The meat replica can be a ground beef replica.

Also provided is a consumable product comprising a comprising a cold setgel comprising one or more isolated and purified proteins from anon-animal source and a salt. In some embodiments, the isolated andpurified plant protein comprises one or more of RuBisCo, moong 8Sglobulin, a pea globulin, a pea albumin, a lentil protein, zein, or anoleosin. In some embodiments, the isolated and purified plant proteincomprises a dehydrin, a hydrophilin, or an intrinsically disorderedprotein. In some embodiments, the cold set gel further comprises a plantderived lipid or microbial derived lipid. In some embodiments, the plantderived lipid or microbial derived lipid is lecithin and/or oil.

The disclosure further provides an adipose tissue replica comprising oneor more isolated plant proteins, one or more plant or algal derivedoils, and optionally a phospholipid. In some embodiments, thephospholipid is lecithin. In some embodiments, the plant based oils areselected from the group consisting of corn oil, olive oil, soy oil,peanut oil, walnut oil, almond oil, sesame oil, cottonseed oil, rapeseedoil, canola oil, safflower oil, sunflower oil, flax seed oil, palm oil,palm kernel oil, coconut oil, babassu oil, shea butter, mango butter,cocoa butter, wheat germ oil, rice bran oil, and combinations thereof.In some embodiments, the fat release temperature of the adipose tissuereplica is between 23° C. to 33° C., 34° C. to 44° C., 45° C. to 55° C.,56° C. to 66° C., 67° C. to 77° C., 78° C. to 88° C., 89° C. to 99° C.,100° C. to 110° C., 111° C. to 121° C., 122° C. to 132° C., 133° C. to143° C., 144° C. to 154° C., 155° C. to 165° C., 166° C. to 167° C.,168° C. to 169° C., 170° C. to 180° C., 181° C. to 191° C., 192° C. to202° C., 203° C. to 213° C., 214° C. to 224° C., 225° C. to 235° C.,236° C. to 246° C., 247° C. to 257° C., 258° C. to 268° C., 269° C. to279° C., 280° C. to 290° C., or 291° C. to 301° C. In some embodiments,the percent fat release of the adipose tissue replica is 0 to 10%, 10%to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70%to 80%, 80% to 90%, or 90% to 100% upon cooking. In some embodiments,the isolated and purified plant protein comprises one or more ofRuBisCo, moong 8S globulin, a pea globulin, a pea albumin, a lentilprotein, zein, or an oleosin.

In some embodiments, the adipose tissue replica comprises from about 40%to about 90% of the oil. In some embodiments, the adipose tissue replicacomprises from about 1% to about 6% of the isolated and purified plantprotein. In some embodiments, the adipose tissue replica comprises fromabout 0.05 to about 2% of the phospholipid. In some embodiments, thefirmness of the adipose tissue replica is similar to that of beefadipose tissue.

Also provided is a consumable product comprising a heme-containingprotein and (i) carbon monoxide and/or (ii) a nitrite, wherein theconsumable product does not comprise meat. In some embodiments, theheme-containing protein accounts for at least 0.01% of the composition.In some embodiments, the consumable product further comprises one ormore ammonium, sodium, potassium, or calcium salts. In some embodiments,the isolated and purified proteins are crosslinked.

Further provided is a consumable product comprising a gelled emulsion,wherein the gelled emulsion comprises:

a) an isolated and purified protein;

b) a first lipid that when not in the consumable product is solid at aselected temperature range; and

c) a second lipid that when not in the consumable product is liquid atthe selected temperature range; wherein the melting temperature of themixture of the first and second lipids is similar to the meltingtemperature of lipids found in meat, and wherein the first and secondlipids are plant derived lipids or microbial derived lipids.

The disclosure also provides a method for making a consumable productcomprising:

a) preparing a solution comprising an isolated and purified plantprotein, wherein the isolated and purified plant protein has (i) asolubility in the solution of at least 25 at a temperature between about2° C. and 32° C., wherein the solution has a pH between 3 and 8, and hasa sodium chloride content of 0 to 300 mM or (ii) a solubility in thesolution of at least 1 mg/ml at a temperature of between 90° C. and 110°C., wherein the solution has a pH between 5 and 8 and has a sodiumchloride content of 0 to 300 mM; and

b) adding the solution to a beverage.

In some embodiments, the solution comprises two or more isolated andpurified plant proteins. In some embodiments, the beverage is clear. Insome embodiments, the isolated and purified plant protein is at aconcentration of at least 1% by weight in the solution. In someembodiments, the isolated and purified plant protein is selected fromthe group consisting of RuBisCo, a moong globulin, a soy globulin, a peaglobulin, a pea albumin, a prolamin, a lentil protein, a dehydrin, ahydrophilin, and an intrinsically disordered protein. In someembodiments, the isolated and purified plant protein is lyophilizedprior to making said solution. In some embodiments, the beverage has animproved mouthfeel compared to a corresponding beverage without theisolated and purified protein.

Also provided is a method for extending the shelf-life of a meat-freeconsumable product, the method comprising adding a heme-containingprotein to the consumable product, wherein the heme containing proteinoxidizes more slowly than myoglobin under equivalent storage conditions.In some embodiments, the heme-containing protein comprises an amino acidsequence with at least 70% homology to an amino acid sequence set forthin any one of SEQ ID NOs: 1-27.

Further provided is a method for making a meat replica comprising a coldset gel, wherein the method includes:

a) denaturing a solution comprising at least one isolated and purifiedprotein from a non-animal source under conditions wherein the isolatedand purified protein does not precipitate out of the solution;

b) optionally adding any heat-labile components to the solution ofdenatured protein;

c) gelling the solution of denatured protein at about 4° C. to about 25°C. by increasing the ionic strength of the solution to form a cold setgel; and

d) incorporating the cold set gel into a meat replica.

In some embodiments, the gelling is induced using 5 to 100 mM sodium orcalcium chloride. In some embodiments, the heat-labile components areproteins or lipids, or mixtures thereof. In some embodiments, theprotein is a heme-containing protein. In some embodiments, the cold setgel is formed in a matrix comprising a freeze-aligned plant protein.

In some embodiments, the isolated and purified protein from a non-animalsource is a plant protein. In some embodiments, the plant protein isselected from the group consisting of RuBisCo, a moong globulin, a soyglobulin, a pea globulin, a pea albumin, a prolamin, a lentil protein, adehydrin, a hydrophilin, and an intrinsically disordered protein.

Further provided is an adipose tissue replica, comprising

a) an isolated and purified non-animal protein;

b) a non-animal lipid; and

c. a three-dimensional matrix comprising fibers derived from non-animalsources, wherein the lipid and the protein are dispersed in thethree-dimensional matrix, and wherein the three-dimensional matrixstabilizes the structure of the adipose tissue replica.

Also provided is a connective tissue replica comprising one or moreisolated and purified proteins assembled into fibrous structures by asolution spinning process. In some embodiments, the fibrous structuresare stabilized by a cross-linking agent.

Provided herein is a method for imparting a beef like flavor to aconsumable product, comprising adding to the consumable composition aheme-containing protein, wherein after cooking, a beef-like flavor isimparted to the consumable composition.

Also provided is a method for making a poultry or a fish compositiontaste like beef, the method comprising adding a heme protein to thepoultry or fish composition, respectively.

In some embodiments, the heme-containing protein has an amino acidsequence with at least 70% homology to any one of the amino acidsequences set forth in SEQ ID NOs: 1-27.

Further provided is a method of making a coacervate, the methodcomprising

a) acidifying a solution of one or more plant proteins to a pH between3.5 and 5.5, wherein the solution comprises 100 mM or less of sodiumchloride; and

b) isolating the coacervate from the solution. In some embodiments, thepH is between 4 and 5. In some embodiments, the plant proteins compriseone or more pea proteins, chickpea proteins, lentil proteins, lupineproteins, other legume proteins, or mixtures thereof. In someembodiments, the pea proteins comprise isolated and purified legumins,isolated and purified vicilins, isolated and purified convicilins, orcombinations thereof. In some embodiments, the isolated and purified peaproteins comprise isolated and purified vicilins and isolated andpurified convicilins. In some embodiments, the acidifying step is donein the presence of a plant derived lipid or microbial derived lipid. Insome embodiments, the plant derived lipid or microbial derived lipidcomprises oils and/or phospholipids.

Provided herein is a method of making an adipose tissue replica, themethod comprising forming an emulsion comprising one or more isolatedplant proteins, one or more plant or algal derived oils, and optionallya phospholipid. In some embodiments, when the phosoholipid is included,it is lecithin. In some embodiments, the plant based oils are selectedfrom the group consisting of corn oil, olive oil, soy oil, peanut oil,walnut oil, almond oil, sesame oil, cottonseed oil, rapeseed oil, canolaoil, safflower oil, sunflower oil, flax seed oil, palm oil, palm kerneloil, coconut oil, babassu oil, shea butter, mango butter, cocoa butter,wheat germ oil, rice bran oil, and combinations thereof. In someembodiments, the fat release temperature of the adipose tissue replicais between 23° C. to 33° C., 34° C. to 44° C., 45° C. to 55° C., 56° C.to 66° C., 67° C. to 77° C., 78° C. to 88° C., 89° C. to 99° C., 100° C.to 110° C., 111° C. to 121° C., 122° C. to 132° C., 133° C. to 143° C.,144° C. to 154° C., 155° C. to 165° C., 166° C. to 167° C., 168° C. to169° C., 170° C. to 180° C., 181° C. to 191° C., 192° C. to 202° C.,203° C. to 213° C., 214° C. to 224° C., 225° C. to 235° C., 236° C. to246° C., 247° C. to 257° C., 258° C. to 268° C., 269° C. to 279° C.,280° C. to 290° C., or 291° C. to 301° C. In some embodiments, thepercent fat release of the adipose tissue replica is 0 to 10%, 10% to20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to80%, 80% to 90%, or 90% to 100% upon cooking. In some embodiments, theisolated and purified plant protein comprises one or more of RuBisCo,moong 8S globulin, a pea globulin, a pea albumin, a lentil protein,zein, or an oleosin. In some embodiments, the emulsion comprises fromabout 40% to about 90% of the oil. In some embodiments, the emulsioncomprises from about 1% to about 4% of the isolated and purified plantprotein. In some embodiments, the adipose tissue replica comprises fromabout 0.05 to about 1% of the phospholipid. In some embodiments, theemulsion is formed by high-pressure homogenization, sonication, or handhomogenization.

Further provided is a method of minimizing undesirable odors or flavorsin a composition comprising plant proteins, the method comprisingcontacting the composition with a ligand having affinity for one or morelipoxygenases.

Also provided is a method of minimizing undesirable odors or flavors ina composition comprising plant proteins, the method comprisingcontacting the composition with activated carbon then removing theactivated carbon from the composition.

Also provided is a method of minimizing undesirable odors or flavors ina composition comprising plant proteins, the method comprising adding alipoxygenase inhibitor and/or an antioxidant to the composition.

The disclosure further provides a chocolate flavored spread comprising:

a) sugar

b) a chocolate flavoring, and

c) a cream fraction from a plant based milk.

Provided herein is a method for altering the texture of a consumableduring or after cooking comprising incorporating within the consumableone or more plant proteins with a low denaturation temperature. In someembodiments, at least one of the one or more plant proteins is isolatedand purified. In some embodiments, the one or more plant proteins areselected from the group consisting of rubisco, pea proteins, lentilproteins, or other legume proteins. In some embodiments, the peaproteins comprise pea albumin proteins. In some embodiments, theconsumable becomes firmer during or after cooking.

Also provided is a tissue replica, which comprises a freeze-alignednon-animal protein. In some embodiments, the non-animal protein is aplant protein. In some embodiments the non-animal protein is isolatedand purified. In some embodiments the tissue replica is a muscle tissuereplica.

The disclosure also provides a meat replica which includes a tissuereplica comprising a freeze-aligned non-animal protein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims. The word “comprising” inthe claims may be replaced by “consisting essentially of” or with“consisting of,” according to standard practice in patent law.

DESCRIPTION OF THE DRAWING

FIG. 1 contains amino acid sequences of exemplary heme-containingproteins.

FIG. 2A is a bar graph depicting the percent fat release based on theamount of lecithin.

FIG. 2B is a bar graph depicting the temperature of the fat releasebased on the amount of lecithin.

FIG. 2C is a bar graph depicting the firmness of adipose replicas basedon the amount of lecithin.

FIG. 3 is a bar graph depicting the percent fat release of adiposereplicas containing different oils (canola oil, cocoa butter, coconutoil, or rice bran oil).

FIG. 4 is a bar graph depicting the fat release temperature of adiposereplicas containing different oils (canola oil, cocoa butter, coconutoil, or rice bran oil).

DETAILED DESCRIPTION I. Consumables

Methods and compositions for producing consumables are described herein.In some cases, the consumables are non-animal based replicas ofanimal-based food products that can be produced by breaking downnon-animal materials into their constituent parts and reassembling thoseparts into the consumables. In certain instances, the consumables arenot intended to replicate an animal-based food and instead have theirown unique characteristics desirable as a food. Additionally, theconsumables may, in some instances, act as nutraceuticals or carriersfor pharmaceutical compositions rather than serving a primary functionas food.

The advantages of the consumables described herein can include, forexample, the using less energy or water in the production of theconsumable compared to similar food products, using no animals in theproduction of the consumable, making a healthier product, using rawmaterials that would otherwise be discarded, or allowing for theelimination (or lack of incorporation) of certain components (e.g.,allergens) from the consumables. The consumables also may have a higherdegree of production consistency, allowing for improved quality controlof the products. Another advantage is that the consumables can bedesigned intentionally to have desirable characteristics for foodpreparation that are superior to traditional food products.

The consumables can be for animal consumption, including humanconsumption. The consumables can be food for domestic animals (e.g., dogfood could be produced according to the present inventions) or wildanimals (e.g., food for non-domesticated predatory animals).

The consumables can be sold in grocery stores, convenience stores, massmerchandisers, and club stores or prepared in restaurants, includingfast food restaurants, schools, event locations, hospitals, militaryfacilities, prisons, shelters, or long-term care facilities, similar toalready existing human foods.

The consumable can be approved by suitable regulatory authorities. Forexample, the consumable could be prepared to be suitable for the U.S.Food and Drug Administration. Methods of the invention can include stepsnecessary to satisfy regulatory agencies.

The consumables of the present invention can replicate, compete with,supplement or replace conventional food products (herein referred to as“food products”). Food products can be any foods which presently exist.The consumables of the invention can be made to replicate the foodproducts, e.g., an equivalent meat product. The equivalent meat productcan be a white meat or a dark meat. The equivalent meat product can bederived from any animal. Non-limiting examples of animals used to derivethe equivalent meat product include farmed animals such as, e.g.,cattle, sheep, pig, chicken, turkey, goose, duck, horse, dog or gameanimals (whether wild or farmed) such as, e.g., rabbit, deer, bison,buffalo, boar, snake, pheasant, quail, bear, elk, antelope, pigeon,dove, grouse, fox, wild pig, goat, kangaroo, emu, alligator, crocodile,turtle, groundhog, marmot, possum, partridge, squirrel, raccoon, whale,seal, ostrich, capybara, nutria, guinea pig, rat, mice, vole, anyvariety of insect or other arthropod, or seafood such as, e.g., fish,crab, lobster, oyster, muscle, scallop, abalone, squid, octopus, seaurchin, tunicate and others.

Many meat products are typically derived from skeletal muscle of ananimal but it is understood that meat can also come from other musclesor organs of the animal. In some embodiments, the equivalent meatproduct is a cut of meat derived from skeletal muscle. In otherembodiments, the equivalent meat product is an organ such as, e.g.,kidney, heart, liver, gallbladder, intestine, stomach, bone marrow,brain, thymus, lung, or tongue. Accordingly, in some embodiments, thecompositions of the present invention are consumables similar toskeletal muscle or organs.

A consumable (e.g., a meat substitute) can comprise one or more of afirst composition comprising a muscle tissue replica, a secondcomposition comprising an adipose tissue replica, and/or a thirdcomposition comprising a connective tissue replica, wherein the one ormore compositions are combined in a manner that recapitulates thephysical organization of meat. The present invention also providesdistinct compositions for a muscle tissue replica (herein referred to as“muscle replica”), an adipose tissue replica (herein referred to as an“adipose replica” or “fat replica”), and a connective tissue replica(herein referred to as “connective tissue replica”). In someembodiments, these compositions are principally or entirely composed ofingredients derived from non-animal sources (e.g., 10% or less of theingredients are from animal sources). In alternative embodiments, themuscle, fat, and/or connective tissue replica, or the meat substituteproducts comprising one or more of the replicas, are partially derivedfrom animal sources but supplemented with ingredients derived fromnon-animal sources. In some embodiments, as much as 90% of the foodproduct is derived from animal sources. In some embodiments about 75% ofthe food product is derived from animal sources. In some embodiments,about 50% of the food product is derived from animal sources. In someembodiments about 10% of the food product is derived from animalsources. In yet other alternative embodiments, the invention providesmeat products substantially derived from animal sources (e.g., a beef,chicken, turkey, or a pork product) that are supplemented with one ormore of a muscle tissue replica, a fat replica, and/or a connectivetissue replica, wherein the replicas are derived substantially orentirely from non-animal sources. A non-limiting example of such a meatproduct is an ultra-lean ground beef product supplemented with anon-animal derived fat replica which improves texture and mouthfeelwhile preserving the health benefits of a consumable low in animal fat.Such alternative embodiments can result in products with properties thatmore closely recapitulate key features associated with preparing andconsuming meat but which are less costly and associated with a lesserenvironmental impact, less animal welfare impact, or improved healthbenefits for the consumer.

Examples of other food products which the consumable can replicate orreplace include: beverages (e.g., cream liquor or milk), protein drinks(e.g., RuBisCo can be used as a protein supplement in beer, distilledalcohol beverages such as vodka, fruit juices, meal replacementbeverages, or water), pastes (e.g. Nutella™, cream, nacho cheese ormayonnaise replicas), pate, blood sausage, meat extenders, eggs, fish,sausage, tenders, spam or chilled foods (e.g., ice cream, yogurt, kefir,sour cream or butter replicas).

The consumables can be a meat replica. The consumables can be made tomimic the cut or appearance of meat. For instance, a consumable may bevisually similar to or indistinguishable from ground beef or aparticular cut of beef. In an example embodiment, the replicas arecombined in a manner that approximates the physical organization ofnatural ground meat (e.g., ground beef, ground chicken, or groundturkey). In other embodiments, the replicas are combined in a mannerthat approximates different cuts of beef, such as, e.g., rib-eye, filetmignon, London broil, among others. Alternatively, the consumables canbe a made with a unique look or appearance. For instance, the consumablecould contain patterns (e.g., lettering or pictures) that are formedfrom the structure of the consumable. In some instances, the consumableslook like traditional food products after they are prepared. Forexample, a consumable may be produced which is larger than a traditionalcut of beef but which, after the consumable is sliced and cooked,appears the same as a traditional cooked meet. In some embodiments theconsumable may resemble a traditional food product shape in twodimensions, but not in a third. For example the consumable may resemblea cut of meat in two dimensions (for example when viewed from the top),but may be much longer (or thicker) than the traditional cut. In thisexample the composition can be cut repeatedly into traditionally meatshaped products.

The consumables can be made from local products. For instance theconsumables can be made from plants grown within a certain radius of theeventual consumer. That radius could be 1, 10, 100, or 1000 miles forexample. Thus, in some embodiments, the invention provides method forproducing a consumable which does not contain products which have beenshipped over 1, 10, 100, or 1000 miles.

The present invention provides methods for producing consistentproperties from the consumables when they are produced from varioussources. For example, a plant based meat replica produced from localplants in Iowa, USA, will have substantially similar taste, odor, andtexture as a plant based meat replica produced from local plants inLorraine, France. This consistency allows for methods for advertisinglocally grown foods with consistent properties. The consistency canarise from the concentration or purification of similar components atdifferent locations. These components can be combined in predeterminedratios to insure consistency. In some embodiments, a high degree ofcharacteristic consistency is possible using components (e.g. isolatedor concentrated proteins and fats) which come from the same plantspecies. In some embodiments, a high degree of characteristicconsistency is possible using components (e.g., isolated or concentratedproteins and fats) which come from the different plant species. In someembodiments, the same proteins can be isolated from different plantspecies (i.e. homologous proteins). In some embodiments, the inventionprovides for a method comprising isolating similar plant constituentsfrom plant sources in different locations, assembling in both locationscompositions provided herein, and selling the compositions, wherein thecompositions assembled and sold at different the geographic locationshave consistent physical and chemical properties. In some embodiments,the isolated constituents are from different plant populations indifferent locations. In some embodiments one or more of the isolatedconstituents are shipped to the separate geographic locations.

The consumables may require fewer resources to produce than consumablesproduced from domesticated animals. Accordingly, the present inventionprovides for meat replicates which require less water or energy toproduce than meat. For example a consumable described herein can requireless than about 10, 50, 100, 200, 300, 500, or 1000 gallons of water perpound of consumable. For comparison producing beef can require over 2000gallons of water per pound of meat.

The consumable may require less land area to produce than a meat productwith similar protein content. For example, a consumable described hereinmay require 30% or less of the land area required to produce a meatproduct with similar protein content.

The consumable may have health benefits compared to an animal product itreplaces in the diet. For example it may have less cholesterol or lowerlevels of saturated fats than comparable meat products. The AmericanHeart Association and the National Cholesterol Education Programrecommends limiting cholesterol intake from food to 300 mg per day,which is equivalent to the consumption of 12 ounces of beef or two eggyolks. Consumables described herein that are indistinguishable to animalproducts such as ground beef, and having a reduced cholesterol contentor no cholesterol, can help maintain a low cholesterol diet. In anotherexample, a consumable described herein may contain no cholesterol, orhigher levels of poly-unsaturated fatty acids compared to the animalproduct it replaces.

The consumable may have animal welfare benefits compared to an animalproduct it replaces in the diet. For instance, it may be producedwithout requiring confinement, forced feeding, premature weaning,disruption of maternal-offspring interactions, or slaughter of animalsfor their meat.

The consumable may have a smaller “carbon footprint” than the meatproducts they replace. For example the consumable may result in netgreenhouse gas emissions of 1%, 5%, 10%, 25%, 50% or 75% of thegreenhouse gas emissions attributable to the animal product it replaces.By way of example, according to the Environmental Working Group (2011)“meat eaters guide to Climate Change and Health,” the production of beefcauses the emission of 27 kg equivalents of carbon dioxide per kilogramof beef that is consumed, and the production of lamb causes the emissionof 39 kg equivalents of carbon dioxide per kilogram of beef that isconsumed.

The consumable described herein may provide alternatives to animalproducts or combinations of animal products whose consumption isforbidden by religious beliefs. For example, the consumable may be akosher replica pork chop.

The consumable can also be shipped in components and produced orassembled at a different location. When available, local components canbe used for production of the consumable. The local components can besupplemented with components that are not locally available. This allowsfor methods of producing consumables, for instance meat replicates,using less energy in shipment than is required for meat. For example,local water can be used in combination with a kit which provides othercomponents of the consumable. Using local water reduces shipping weight,thereby reducing cost and environmental impact.

The consumables described herein can be produced or assembled wholly orin part in areas where animal farming is not practical or is notallowed. The consumable can be produced or assembled within an urbanenvironment. For example, a kit may be provided to a user to enable theuser to produce the consumable. The user could use local water or useplants from a rooftop garden, for instance in Shanghai. In anotherexample, the consumables could be produced aboard a space craft, spacestation, or lunar base. Accordingly, the present invention providesmethods and systems for the production of meat replicas for use in spacetravel or for training for the same. For instance the present inventioncould be used in earth based training for space travel. The consumablescould also be produced on an island or upon a manmade platform at seawhere the keeping of livestock is difficult or prohibited.

II. Properties of the Consumable

The consumables described herein are typically designed to replicate theexperience of eating a food product, e.g., meat. The look, texture, andtaste of the consumable can be such that it is similar to, orindistinguishable from, a food product, e.g., meat. The consumable canalso be produced to have the desirable characteristics of food productswithout incorporating other undesirable characteristics. For example, aconsumable can be a replica steak that does not have gristle or othercomponents not typically consumed in a predicate food product.

The invention provides, in certain embodiments, methods for determiningthe suitability for a consumable to qualify as a replica of a foodproduct, for example, by determining whether an animal or human candistinguish the consumable from a predicate food product, e.g., aparticular meat. One method to determine whether the consumable iscomparable to a food product (e.g. meat) is to a) define the propertiesof meat and b) determine whether the consumable has similar properties.

Properties that can be tested or used to compare or describe a foodproduct or the consumable include mechanical properties such ashardness, cohesiveness, brittleness, chewiness, gumminess, viscosity,elasticity, and adhesiveness. Properties of food products that can betested also include geometric properties such as particle size andshape, and particle shape and orientation. The three dimensionalorganization of particles may also be tested. Additional properties caninclude moisture content and fat content. These properties can bedescribed using terms such as “soft,” “firm” or “hard” describehardness; “crumbly,” “crunchy,” “brittle,” “chewy,” “tender,” “tough,”“short,” “mealy,” “pasty,” or “gummy,” to describe cohesiveness; “thin”or “viscous” to describe viscosity; “plastic” or “elastic” to describeelasticity; “sticky,” “tacky” or “gooey” to describe adhesiveness;“gritty,” “grainy” or “course” to describe particle shape and size;“fibrous,” “cellular” or “crystalline” to describe particle shape andorientation, “dry,” “moist,” “wet,” or “watery” to describe moisturecontent; or “oily” or “greasy” to describe fat content. Accordingly, inone embodiment, a group of people can be asked to rate a certain foodproduct, for instance ground beef, according to properties whichdescribe the food product. A consumable described herein can be rated bythe same people to determine equivalence.

Flavor of the food product can also be assessed. Flavors can be ratedaccording to similarity to food products, e.g., “eggy,” “fishy,”“buttery,” “chocolaty,” “fruity”, “peppery,” “baconlike,” “creamy,”“milky,” “or “beefy.” Flavors can be rated according to the seven basictastes, i.e., sweet, sour, bitter, salty, umami (savory), pungent (orpiquant), and metallic. Flavors can be described according to thesimilarity to an experience caused by a chemical, e.g., diacetyl(buttery), 3-hydroxy-2 butanone (buttery), nona-2E-enal (fatty),1-octene-3-ol (mushroom), hexanoic acid (sweaty), 4-hydroxy-5-methylfuranone (HMF, meaty), pyrazines (nutty), bis(2-methyl-3-furyl)disulfide (roast meat), decanone (musty/fruity), isoamyl acetate(banana), benzaldehyde (bitter almond), cinnamic aldehyde (cinnamon),ethyl propionate (fruity), methyl anthranilate (grape), limonene(orange), ethyl decadienoate (pear), allyl hexanoate (pineapple), ethylmaltol (sugar, cotton candy), ethylvanillin (vanilla), butanoic acid(rancid), 12-methyltridecanal (beefy), or methyl salicylate(wintergreen). These ratings can be used as an indication of theproperties of the food product. The consumables of the present inventioncan then be compared to the food product to determine how similar theconsumable is to the food product. In some instances the properties ofthe consumables are then altered to make the consumable more similar tothe food product. Accordingly, in some embodiments, the consumable israted similar to a food product according to human evaluation. In someembodiments the consumable is indistinguishable from real meat to ahuman.

The consumables can be made to eliminate properties associated with thesource of the components of the consumables. For example a consumablecan be made from components obtained from beans but can be made to lacka “beany” flavor or texture. One way this can be achieved is by breakingdown the component source materials into isolated and purifiedcomponents and not using components that cause undesired characteristicproperties of the source. In addition, as described herein, off-flavorsor aromas (e.g., undesirable flavors or aromas) in isolated and/orpurified components can be minimized by deodorizing with activatedcharcoal or by removing enzymes such as lipoxygenases (LOX), which canbe present in trace amounts and which can convert unsaturatedtriacylglycerides (such as linoleic acid or linolenic acid) into smallerand more volatile molecules. LOX are naturally present in legumes suchas peas, soybeans, and peanuts, as well as rice, potatoes, and olives.When legume flours are fractionated into separate protein fractions, LOXcan act as undesirable “time-bombs” that can cause undesirable flavorsor aromas on aging or storage. As shown in Example 34, compositionscontaining plant proteins (e.g., from ground plant seeds) can besubjected to purification to remove LOX using, for example, an affinityresin that binds to LOX and removes it from the protein sample. Theaffinity resin can be linoleic acid, linolenic acid, stearic acid, oleicacid, propyl gallate, or epigalloccatechin gallate attached to a solidsupport such as a bead or resin. See, e.g., WO2013138793. In addition,depending on the protein component, certain combinations of antioxidantsand/or LOX inhibitors can be used as effective agents to minimizeoff-flavor or off-odor generation in protein solutions, especially inthe presence of fats and oils. Such compounds can include, for example,one or more of β-carotene, α-tocopherol, caffeic acid, propyl gallate,or epigallocatechin gallate. These may be included during purificationof the proteins or during subsequent food processing steps to mitigategeneration of off-flavors or off-odors in protein-based foods.

In some compositions, subjects asked to identify the consumable identifyit as a form of a food product, or as a particular food product, e.g., asubject will identify the consumable as meat. For example, in somecompositions a human will identify the consumable as having propertiesequivalent to meat. In some embodiments one or more properties of theconsumable are equivalent to the corresponding properties of meataccording to a human's perception. Such properties include theproperties that can be tested. In some embodiments a human identifies aconsumable of the present invention as more meat like than any meatsubstitutes found in the art.

Experiments can demonstrate that a consumable is acceptable toconsumers. A panel can be used to screen a variety of consumablesdescribed herein. A number of human panelists can test multipleconsumable samples, namely, natural meats vs. the consumablecompositions described herein, or a meat substitute vs. a consumablecomposition described herein. Variables such as fat content can bestandardized, for example to 20% fat using lean and fat meat mixes. Fatcontent can be determined using the Babcock for meat method (S. S.Nielson, Introduction to the Chemical Analysis of Foods (Jones &Bartlett Publishers, Boston, 1994)). Mixtures of ground beef andconsumables of the invention prepared according to the proceduredescribed herein can be formulated.

Panelists can be served samples (e.g., in booths), under red lights orunder white light, in an open consumer panel. Samples can be assignedrandom three-digit numbers and rotated in ballot position to preventbias. Panelists can be asked to evaluate samples for tenderness,juiciness, texture, flavor, and overall acceptability using a hedonicscale from 1=dislike extremely, to 9=like extremely, with a median of5=neither like nor dislike. Panelists can be encouraged to rinse theirmouths with water between samples, and given opportunity to comment oneach sample.

The results of this experiment can indicate significant differences orsimilarities between the traditional meats and the compositions of theinvention.

These results can demonstrate that the compositions described herein arejudged as acceptably equivalent to real meat products. Additionally,these results can demonstrate that compositions described herein arepreferred by panelist over other commercially available meatsubstitutes. Thus, in some embodiments the present invention providesfor consumables that are similar to traditional meats and are more meatlike than previously known meat alternatives.

Consumables of the invention can also have similar physicalcharacteristics as food products, e.g., traditional meat. In oneembodiment, the force required to pierce a 1 inch thick structure (e.g.,a patty) made of a consumable of the invention with a fixed diametersteel rod is not significantly different than the force required topierce a 1 inch thick similar food product structure (e.g., a groundbeef patty) with a similar fixed diameter steel rod. Accordingly, theinvention provides for consumables with similar physical strengthcharacteristics to meat. In another embodiment, the force required totear a sample of the invention with a cross-sectional area of 100 mm² isnot significantly different than the force required to tear a sample ofanimal tissue (muscle, fat or connective tissue) with a cross-sectionalarea 100 mm² measured the same way. Force can be measured using, forexample, TA.XT Plus Texture Analyzer (Textrue Technologies Corp.).Accordingly, the invention provides for consumables with similarphysical strength characteristics to meat.

Consumables described herein can have a similar cook loss characteristicas a food product, e.g., meat. For example a consumable can have asimilar fat and protein content to ground beef and have the samereduction in size when cooked as real ground beef. Similarities in sizeloss profiles can be achieved for various compositions of consumablesdescribed herein matched to various meats. The cook loss characteristicsof the consumable also can be engineered to be superior to foodproducts. For example a consumable can be produced that has less lossduring cooking but achieves similar tastes and texture qualities as thecooked products. One way this is achieved is by altering the proportionsof lipids based on melting temperatures in the consumable composition.Another way this is achieved is by altering the protein composition ofthe consumable by controlling the concentration of protein or by themechanism by which the tissue replica is formed.

In some embodiments, the consumable is compared to an animal based foodproduct (e.g., meat) based upon olfactometer readings. In variousembodiments the olfactometer can be used to assess odor concentrationand odor thresholds, or odor suprathresholds with comparison to areference gas, hedonic scale scores to determine the degree ofappreciation, or relative intensity of odors. In some embodiments, theolfactometer allows the training and automatic evaluation of expertpanels. So in some embodiments the consumable is a product that causessimilar or identical olfactometer readings. In some embodiments thedifferences are sufficiently small to be below the detection thresholdof human perception.

Gas chromatography-mass spectrometry (GCMS) is a method that combinesthe features of gas-liquid chromatography and mass spectrometry toseparate and identify different substances within a test sample. GCMScan, in some embodiments, be used to evaluate the properties of aconsumable. For example volatile chemicals can be isolated from the headspace around meat. These chemicals can be identified using GCMS. Aprofile of the volatile chemicals in the headspace around meat isthereby created. In some instances each peak of the GCMS can be furtherevaluated. For instance, a human could rate the experience of smellingthe chemical responsible for a certain peak. This information could beused to further refine the profile. GCMS could then be used to evaluatethe properties of the consumable. The GCMS profile can be used to refinethe consumable.

Characteristic flavor and fragrance components are mostly producedduring the cooking process by chemical reactions molecules includingamino acids, fats and sugars which are found in plants as well as meat.Therefore, in some embodiments, the consumable is tested for similarityto meat during or after cooking. In some embodiments human ratings,human evaluation, olfactometer readings, or GCMS measurements, orcombinations thereof, are used to create an olfactory map of cookedmeat. Similarly, an olfactory map of the consumable, for instance a meatreplica, can be created. These maps can be compared to assess howsimilar the cooked consumable is to meat. In some embodiments theolfactory map of the consumable during or after cooking is similar to orindistinguishable from that of cooked or cooking meat. In someembodiments the similarity is sufficient to be beyond the detectionthreshold of human perception. The consumable may be created so itscharacteristics are similar to a food product after cooking, but theuncooked consumable may be have properties that are different from thepredicate food product prior to cooking.

Shelf life is the length of time that consumables are given before theyare considered unsuitable for sale, use, or consumption. Generally, itis important to maintain a meat product at about 2° C. as the shelf lifedecreases with exposure to higher temperatures.

The shelf life of meat is determined through research into the meatproducts' sensory cues over time (odor, visual appearance of thepackage, color, taste and texture), and through laboratory analysisunder controlled conditions to determine how long a product remainssafe, wholesome and enjoyable. Ground beef is being used as an example,but similar conditions would apply to steaks, chops and roasts fromother meat types. Beef in its natural state is dark bluish-purple.However, oxygen can permeate into the meat and cause a chemical reactionwith the myoglobin in meat, leading to a red color. Ongoing exposure tooxygen causes oxidation of myoglobin and causes red meat to become brownand develop “off” flavors. To control this oxidation, there has beensignificant research into different methods of storing and displayingmeat products to increase the shelf life of meat products. These includethe use of vacuum packing, modified atmosphere packing (high oxygen),modified atmosphere packaging (low oxygen with carbon monoxide), and/orHigh Pressure Pasteurization (HPP).

The main determinant of the color of meat is the concentration of ironcarrying proteins in the meat. In the skeletal muscle component of meatproducts, one of the main iron-carrying proteins is myoglobin. It isestimated that the white meat of chicken has under 0.05% myoglobin; porkand veal have 0.1-0.3% myoglobin; young beef has 0.4-1.0% myoglobin; andold beef has 1.5-2.0% myoglobin. Normally, myoglobin in meat exists inthree states: Oxymyoglobin (Fe²⁺) (oxygenated=bright red); myoglobin(Fe²⁺) (non-oxygenated=purplish/magenta); and metmyoglobin (Fe²⁺)(oxidized=brown). The transition of oxymyoglobin to metmyoglobin in thepresence of oxygen is thought to be the cause of the color change ofground meat from red to brown. Meat shelf life extenders have beendeveloped to extend the lifetime of the red color of meat productsincluding but not limited to carbon monoxide, nitrites, sodiummetabisulfite, Bombal, vitamin E, rosemary extract, green tea extract,catechins and other anti-oxidants.

However an intrinsically more stable heme protein such a hemoglobinisolated from Aquifex aeolicus (SEQ ID NO:3) or Methylacidiphiluminfernorum (SEQ ID NO: 2) will oxidize more slowly than a mesophilichemoglobin such as myoglobin. The heme proteins described herein (see,e.g., FIG. 1) also may have the lifetime of the reduced heme-Fe²⁺ stateextended by meat shelf life extenders such as carbon monoxide and sodiumnitrite. Heme proteins may be selected for the desired color retentionproperties. For example for low temperature sous-vide cooking, arelatively unstable heme protein such as one from Hordeum vulgare mayprovide a brown product that appears cooked under conditions whereinmyoglobin would retain its red, uncooked appearance. In some embodimentsthe heme protein may be selected to have increased stability where forexample the meat replica may retain an attractive medium rare appearancedespite being thoroughly cooked for food safety.

The main determinant of rancidity and production of off flavors or offodors is the oxidation of components of the consumable, including butnot limited to, the fats. For example, oxidation of unsaturated fattyacids is a known cause of rancid odors. In some embodiments, meatreplicas have extended shelf life because the makeup of the chemicalproperties of the meat replica are controlled such that the taste,texture, smell, and chemical properties do not react with oxygen tocreate off flavors or off odors. In some embodiments the meat replicasare less sensitive to oxidation due to the presence of higher degree ofunsaturated fatty acids than present in beef. In some embodiments themeat replica contains no unsaturated fatty acids. In other embodimentsthe meat replica contains higher levels of anti-oxidants such asglutathione, vitamin C, vitamin A, and vitamin E as well as enzymes suchas catalase, superoxide dismutase and various peroxidases than arepresent in meat. In other embodiments, off flavor or off odor generatingcomponents such as lipoxygenase are not present.

In some embodiments, a consumable described herein shows increasedstability under commercial packaging conditions. In some embodiments,the improved shelf life is improved by using components with increasedoxidative stability such as lipids with reduced levels of unsaturatedfatty acids, and/or by using a more stable heme protein such ahemoglobin isolated from Aquifex aeolicus (SEQ ID NO:3) orMethylacidiphilum infernorum (SEQ ID NO: 2). In some embodiments, theimproved shelf life is due to the combination of components used in theconsumable. In some embodiments, the consumable is designed specificallyfor the desired packaging method.

III. Composition of the Consumables

A consumable described herein includes one or more isolated and purifiedproteins. “Isolated and purified protein” refers to a preparation inwhich the cumulative abundance by mass of protein components other thanthe specified protein, which can be a single monomeric or multimericprotein species, is reduced by a factor of 2 or more, 3 or more, 5 ormore, 10 or more, 20 or more, 50 or more, 100 or more or 1000 or morerelative to the source material from which the specified protein wasisolated. For clarity, the isolated and purified protein is described asisolated and purified relative to its starting material (e.g., plants orother non-animal sources). In some embodiments, the term “isolated andpurified” can indicate that the preparation of the protein is at least60% pure, e.g., greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%pure. The fact that a consumable may comprise materials in addition tothe isolated and purified protein does not change the isolated andpurified nature of the protein as this definition typically applies tothe protein before addition to the composition.

In some embodiments, the one or more isolated and purified proteinsaccounts for at least 1%, at least 5%, at least 10%, at least 20%, atleast 30%, at least 40%, or at least 50% of the protein content of theconsumable by weight. In some embodiments, each of the one or moreisolated proteins is isolated and purified separately.

A consumable described herein may be substantially or entirely composedof ingredients derived from non-animal sources, e.g., plant, fungal, ormicrobial based sources. The plant sources can be organically grownsources. Proteins can be extracted from the source material (e.g.,extracted from animal tissue, or plant, fungal, algal, or bacterialbiomass, or from the culture supernatant for secreted proteins) or froma combination of source materials (e.g., multiple plant species). Theconsumables also can be made from a combination of plant based andanimal based sources. For instance, the consumable may be a ground beefproduct supplemented with plant based products of the invention.

A. Sources of Components of the Consumable

As described above, isolated and purified proteins can be derived fromnon-animal sources such as plants, algae, fungi (e.g., yeast orfilamentous fungi), bacteria, or Archaea. In some embodiments, theisolated and purified proteins can be obtained from genetically modifiedorganisms such as genetically modified bacteria or yeast. In someembodiments, the isolated and purified proteins are chemicallysynthesized or obtained via in vitro synthesis.

In some embodiments, the one or more isolated and purified proteins arederived from plant sources. The isolated and purified proteins can beisolated from a single plant source or, alternatively, multiple plantsources can serve as the starting material for the isolation andpurification of proteins. As described herein, isolated and purifiedplant proteins are soluble in solution. The solution can comprise EDTA(0-0.1M), NaCl (0-1M), KCl (0-1M), NaSO₄ (0-0.2M), potassium phosphate(0-1M), sodium citrate (0-1M), sodium carbonate (0-1M), sucrose (0-50%),Urea (0-2M) or any combination thereof. The solution can have a pH of 3to 11. In some embodiments, plant proteins can have a solubility in asolution of >25 g/L (e.g., at least 25, 30, 35, 40, 45, 50, 75, 100,125, 150, 175, 200, or 225 g/L) at a temperature between about 2° C. andabout 32° C. (e.g., between 3° C. and 8° C., 10° C. and 25° C., or 18°C. and 25° C.), wherein the solution has a pH between 3 and 8 (e.g., pHof 3-6, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8), and has a sodium chloridecontent of 0 to 300 mM (e.g., 50, 100, 150, 200, 250, or 300 mM). Insome embodiments, the isolated and purified proteins are soluble insolution at greater than 10, 15, 20, 25, 50, 100, 150, 200, or 250 g/L.

One of skill in the art will understand that proteins that can beisolated from any organism in the plant kingdom may be used to producethe consumables described herein. Non-limiting examples of plant sourcesinclude grain crops such as, e.g., maize, oats, rice, wheat, barley,rye, millet, sorghum, buckwheat, amaranth, quinoa, triticale (a wheatrye hybrid), teff (Eragrostis tef); oilseed crops including cottonseed,sunflower seed, safflower seed, Crambe, Camelina, mustard, rapeseed(Brassica napus); Acacia, or plants from the legume family, such as,e.g., clover, Stylosanthes, Sesbania, vetch (Vicia), Arachis,Indigofera, Leucaena, Cyamopsis, peas such as cowpeas, english peas,yellow peas, or green peas, or beans such as, e.g., soybeans, favabeans, lima beans, kidney beans, garbanzo beans, mung beans, pintobeans, lentils, lupins, mesquite, carob, soy, and peanuts (Arachishypogaea); leafy greens such as, e.g., lettuce, spinach, kale, collardgreens, turnip greens, chard, mustard greens, dandelion greens,broccoli, or cabbage; or green matter not ordinarily consumed by humans,including biomass crops such as switchgrass (Panicum virgatum),Miscanthus, Arundo donax, energy cane, Sorghum, or other grasses,alfalfa, corn stover, kelp or other seaweeds, green matter ordinarilydiscarded from harvested plants, sugar cane leaves, leaves of trees,root crops such as cassava, sweet potato, potato, carrots, beets, orturnips; or coconut.

Protein can be isolated from any portion of the plant, including theroots, stems, leaves, flowers, or seeds. For example,ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) can beisolated from, for example, alfalfa, carrot tops, corn stover, sugarcane leaves, soybean leaves, switchgrass, Miscanthus, energy cane,Arundo donax, seaweed, kelp, algae or mustard greens.

Proteins that are abundant in plants can be isolated in large quantitiesfrom one or more source plants and thus are an economical choice for usein any of compositions provided herein (e.g., the muscle, fat, orconnective tissue replicas, meat substitute products or others).Accordingly, in some embodiments, the one or more isolated and purifiedproteins comprise an abundant protein found in high levels in a plantand capable of being isolated and purified in large quantities. In someembodiments, the abundant protein comprises about 0.5%, 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, or 70% of the total protein content of the source plantmaterial. In some embodiments, the abundant protein comprises about0.5-10%, about 5-40%, about 10-50%, about 20-60%, or about 30-70% of thetotal protein content of the source plant material. In some embodiments,the abundant protein comprises about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the totalweight of the dry matter of the source plant material. In someembodiments, the abundant protein comprises about 0.5-5%, about 1-10%,about 5-20%, about 10-30%, about 15-40%, or about 20-50% of the totalweight of the dry matter of the source plant material.

The one or more isolated and purified proteins can comprise an abundantprotein that is found in high levels in the leaves of plants. In someembodiments, the abundant protein comprises about 0.5%, 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, or 80% of the total protein content of the leaves ofthe source plant. In some embodiments, the abundant protein comprisesabout 0.5-10%, about 5%-40%, about 10%-60%, about 20%-60%, or about30-70% of the total protein content of the leaves of the source plant.In some embodiments, the one or more isolated proteins comprise RuBisCo,which is a particularly useful protein for meat replicas because of itshigh solubility and an amino acid composition that is close to theoptimum proportions of essential amino acids for human nutrition. Inparticular embodiments, the one or more isolated proteins compriseribulose-1,5-bisphosphate carboxylase oxygenase activase (RuBisCoactivase). In some embodiments, the one or more isolated and purifiedproteins comprise a vegetative storage protein (VSP).

The one or more isolated proteins can comprise an abundant protein thatis found in high levels in the seeds of plants. In some embodiments, theabundant protein comprises about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85% or 90% or more of the total protein content of the seedsof the source plant. In some embodiments, the abundant protein comprisesabout 0.5-10%, about 5%-40%, about 10%-60%, about 20%-60%, or about30-70% or >70% of the total protein content of the seeds of the sourceplant. Non-limiting examples of proteins found in high levels in theseeds of plants include seed storage proteins, e.g., albumins,glycinins, conglycinins, legumins, globulins, vicilins, conalbumin,gliadin, glutelin, gluten, glutenin, hordein, prolamins, phaseolin(protein), proteinoplast, secalin, triticeae gluten, or zein, or oilbody proteins such as oleosins, caloleosins, or steroleosins.

The one or more isolated and purified proteins can include highlysoluble proteins such as dehydrins, hydrophilins, natively unfoldedproteins (also referred to as intrinsically disordered proteins), orother proteins of the late-embryogenesis abundant (LEA) family. LEAproteins have been found in animals, plants and microorganisms and arethought to act as osmoprotectants and stress response proteins. See,e.g., Battaglia, et al., Plant Physiol., 148:6-24 (2008). Such proteinsalso are heat stable. Such LEA proteins can have a solubility in asolution of at least 1 g/L (e.g., 2, 4, 6, 8, 10, 15, 20, 25, 50, 100,150, 200, or 250 g/L) at a temperature of between 90° C. and 110° C.(e.g., between 95° C. and 105° C., 95° C., or 100° C.), wherein thesolution has a pH between 5 and 8 (e.g., pH of 5, 5.5, 6, 6.5, 7, 7.5,or 8) and has a sodium chloride content of 0 to 300 mM (e.g., 50, 100,150, 200, 250, or 300 mM). In some cases, the LEA proteins may beisolated by heating a protein extract to 90° C.-110° C. (e.g., 95° C. or100°) and, after centrifugation or filtration of insoluble material,concentrating the LEA protein fraction by, for example, ultrafiltration.In some cases, isoionic pH precipitation, trichloroacetic acidprecipitation, and/or ammonium sulfate precipitation steps can be donebefore or after the heating step to additionally remove non-LEAproteins. Heating the solution to 90° C.-110° C. denatures mostproteins, allows the majority of the proteins to be removed fromsolution.

B. Proteins

Without being bound by theory, it is believed that by isolating andpurifying non-animal proteins (e.g., plant proteins), consumables can bemade with greater consistency and greater control over the properties ofthe consumable. In some embodiments, about 0.1%, 0.2%, 0.5%, 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the proteincomponent of the consumable is comprised of one or more isolated andpurified proteins. The isolated and purified protein may be greater than60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% pure.

The isolated and purified proteins can be isolated from one or moreother components of a non-animal source. For example, a protein fractionmay be isolated from an isolate of a plant. The isolated proteins may insome cases be purified, wherein a certain kind of protein is separatedfrom other components found in the non-animal source. Proteins can beseparated on the basis of their molecular weight, for example, by sizeexclusion chromatography, ultrafiltration through membranes, or densitycentrifugation. In some embodiments, the proteins can be separated basedon their surface charge, for example, by isoelectric precipitation,anion exchange chromatography, or cation exchange chromatography.Proteins also can be separated on the basis of their solubility, forexample, by ammonium sulfate precipitation, isoelectric precipitation,surfactants, detergents or solvent extraction. Proteins also can beseparated by their affinity to another molecule, using, for example,hydrophobic interaction chromatography, reactive dyes, orhydroxyapatite. Affinity chromatography also can include usingantibodies having specific binding affinity for the protein of interest,nickel NTA for His-tagged recombinant proteins, lectins to bind to sugarmoieties on a glycoprotein, or other molecules which specifically bindsthe protein of interest.

Isolating proteins allows for the elimination of unwanted material. Insome embodiments, an isolated and purified protein is a protein that hasbeen substantially separated from unwanted material (e.g., nucleic acidssuch as RNA and DNA, lipid membranes, phospholipids, fats, oils,carbohydrates such as starch, cellulose, and glucans, phenoliccompounds, polyphenolic compounds, aromatic compounds, or pigments) inthe seeds, leaves, stems, or other portion of the plant.

The isolated and purified proteins also can be recombinantly producedusing polypeptide expression techniques (e.g., heterologous expressiontechniques using bacterial cells, insect cells, fungal cells such asyeast cells, plant cells, or mammalian cells). In some cases, standardpolypeptide synthesis techniques (e.g., liquid-phase polypeptidesynthesis techniques or solid-phase polypeptide synthesis techniques)can be used to produce proteins synthetically. In some cases, cell-freetranslation techniques can be used to produce proteins synthetically.

The protein or proteins incorporated into the consumable can serve anutritional function. In some instance, the protein also serves to alterthe properties of the consumable, e.g., the flavor, color, odor, and/ortexture of the consumable. For example, a meat substitute product cancomprise a protein indicator that indicates cooking progression from araw state to a cooked state, wherein the meat substitute product isderived from non-animal sources.

Examples of proteins that can be isolated and purified, and used in theconsumables described herein include ribosomal proteins, actin,hexokinase, lactate dehydrogenase, fructose bisphosphate aldolase,phosphofructokinases, triose phosphate isomerases, phosphoglyceratekinases, phosphoglycerate mutases, enolases, pyruvate kinases,proteases, lipases, amylases, glycoproteins, lectins, mucins,glyceraldehyde-3-phosphate dehydrogenases, pyruvate decarboxylases,actins, translation elongation factors, histones,ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCo),ribulose-1,5-bisphosphate carboxylase oxygenase activase (RuBisCoactivase), albumins, glycinins, conglycinins, globulins, vicilins,conalbumin, gliadin, glutelin, gluten, glutenin, hordein, prolamin,phaseolin (protein), proteinoplast, secalin, extensins, triticeaegluten, collagens, zein, kafirin, avenin, dehydrins, hydrophilins, lateembyogenesis abundant proteins, natively unfolded proteins, any seedstorage protein, oleosins, caloleosins, steroleosins or other oil bodyproteins, vegetative storage protein A, vegetative storage protein B,moong seed storage 8S globulin, globulin, pea globulins, and peaalbumins.

In some embodiments, an isolated and purified protein can be a proteinthat interacts with lipids and helps to stabilize lipids in a structure,a protein that binds lipids and helps crosslink lipid structures, or aprotein that binds lipids and helps crosslink lipid structures andnon-lipid interacting proteins. Without wishing to be bound by aparticular theory, using such proteins in a consumable described hereinmay improve the integration of lipids and/or fat replicas with othercomponents of the meat substitute product, resulting in improvedmouthfeel and texture of the final product. A non-limiting example of alipid-interacting plant protein includes proteins in the oleosin family.Oleosins are lipid-interacting proteins that are found in oil bodies ofplants. Other non-limiting examples of plant proteins that can interactwith lipids and stabilize emulsions include seed storage proteins fromGreat Northern beans, albumins from peas, globulins from peas, 8Sglobulins from moong bean, 8S globulins from Kidney bean, prolamin andlipid transfer proteins.

In some embodiments, one or more of the isolated and purified proteinscan be an iron-carrying protein such as a heme-containing protein. Asused herein, the term “heme containing protein” can be usedinterchangeably with “heme containing polypeptide” or “heme protein” or“heme polypeptide” and includes any polypeptide that can covalently ornoncovalently bind a heme moiety. In some embodiments, theheme-containing polypeptide is a globin and can include a globin fold,which comprises a series of seven to nine alpha helices. Globin typeproteins can be of any class (e.g., class I, class II, or class III),and in some embodiments, can transport or store oxygen. For example, aheme-containing protein can be a non-symbiotic type of hemoglobin or aleghemoglobin. A heme-containing polypeptide can be a monomer, i.e., asingle polypeptide chain, or can be a dimer, a trimer, tetramer, and/orhigher order oligomers. The life-time of the oxygenated Fe²⁺ state of aheme-containing protein can be similar to that of myoglobin or canexceed it by 10%, 20%, 30% 50%, 100% or more under conditions in whichthe heme-protein-containing consumable is manufactured, stored, handledor prepared for consumption. The life-time of the unoxygenated Fe²⁺state of a heme-containing protein can be similar to that of myoglobinor can exceed it by 10%, 20%, 30% 50%, 100% or more under conditions inwhich the heme-protein-containing consumable is manufactured, stored,handled or prepared for consumption

Non-limiting examples of heme-containing polypeptides can include anandroglobin, a cytoglobin, a globin E, a globin X, a globin Y, ahemoglobin, a leghemoglobin, a flavohemoglobin, Hell's gate globin I, amyoglobin, an erythrocruorin, a beta hemoglobin, an alpha hemoglobin, aprotoglobin, a cyanoglobin, a cytoglobin, a histoglobin, a neuroglobins,a chlorocruorin, a truncated hemoglobin (e.g., HbN or HbO), a truncated2/2 globin, a hemoglobin 3 (e.g., Glb3), a cytochrome, or a peroxidase.

Heme-containing proteins that can be used in the consumables describedherein can be from mammals (e.g., farms animals such as cows, goats,sheep, horses, pigs, ox, or rabbits), birds, plants, algae, fungi (e.g.,yeast or filamentous fungi), ciliates, or bacteria. For example, aheme-containing protein can be from a mammal such as a farm animal(e.g., a cow, goat, sheep, pig, ox, or rabbit) or a bird such as aturkey or chicken. Heme-containing proteins can be from a plant such asNicotiana tabacum or Nicotiana sylvestris (tobacco); Zea mays (corn),Arabidopsis thaliana, a legume such as Glycine max (soybean), Cicerarietinum (garbanzo or chick pea), Pisum sativum (pea) varieties such asgarden peas or sugar snap peas, Phaseolus vulgaris varieties of commonbeans such as green beans, black beans, navy beans, northern beans, orpinto beans, Vigna unguiculata varieties (cow peas), Vigna radiata (Mungbeans), Lupinus albus (lupin), or Medicago sativa (alfalfa); Brassicanapus (canola); Triticum sps. (wheat, including wheat berries, andspelt); Gossypium hirsutum (cotton); Oryza sativa (rice); Zizania sps.(wild rice); Helianthus annuus (sunflower); Beta vulgaris (sugarbeet);Pennisetum glaucum (pearl millet); Chenopodium sp. (quinoa); Sesamum sp.(sesame); Linum usitatissimum (flax); or Hordeum vulgare (barley).Heme-containing proteins can be isolated from fungi such asSaccharomyces cerevisiae, Pichia pastoris, Magnaporthe oryzae, Fusariumgraminearum, or Fusarium oxysporum. Heme-containing proteins can beisolated from bacteria such as Escherichia coli, Bacillus subtilis,Bacillus megaterium, Synechocistis sp., Aquifex aeolicus,Methylacidiphilum infernorum, or thermophilic bacteria (e.g, that growat temperatures greater than 45° C.) such as Thermophilus.Heme-containing proteins can be isolated from algae such asChlamydomonas eugametos. Heme-containing proteins can be isolated fromprotozoans such as Paramecium caudatum or Tetrahymena pyriformis. Insome embodiments, the bacterial hemoglobins are selected from the groupconsisting of Aquifex aeolicus, Thermobifida fusca, Methylacidiphiluminfernorum (Hells Gate), Synechocystis SP, or Bacillus subtilis. Thesequences and structure of numerous heme-containing proteins are known.See for example, Reedy, et al., Nucleic Acids Research, 2008, Vol. 36,Database issue D307-D313 and the Heme Protein Database available on theworld wide web at http://hemeprotein.info/heme.php.

For example, a non-symbiotic hemoglobin can be from a plant selectedfrom the group consisting of soybean, sprouted soybean, alfalfa, goldenflax, black bean, black eyed pea, northern, garbanzo, moong bean,cowpeas, pinto beans, pod peas, dried peas, quinoa, sesame, sunflower,wheat berries, spelt, barley, wild rice, or rice.

Any of the heme-containing proteins described herein that can be usedfor producing consumables can have at least 70% (e.g., at least 75%,80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to theamino acid sequence of the corresponding wild-type heme-containingprotein or fragments thereof that contain a heme-binding motif. Forexample, a heme-containing protein can have at least 70% sequenceidentity to an amino acid sequence set forth in FIG. 1, including anon-symbiotic hemoglobin such as that from Vigna radiata (SEQ ID NO:1),Hordeum vulgare (SEQ ID NO:5), Zea mays (SEQ ID NO:13), Oryza sativasubsp. japonica (rice) (SEQ ID NO:14), or Arabidopsis thaliana (SEQ IDNO:15), a Hell's gate globin I such as that from Methylacidiphiluminfernorum (SEQ ID NO:2), a flavohemoprotein such as that from Aquifexaeolicus (SEQ ID NO:3), a leghemoglobin such as that from Glycine max(SEQ ID NO:4), Pisum sativum (SEQ ID NO:16), or Vigna unguiculata (SEQID NO:17), a heme-dependent peroxidase such as from Magnaporthe oryzae,(SEQ ID NO:6) or Fusarium oxysporum (SEQ ID NO:7), a cytochrome cperoxidase from Fusarium graminearum (SEQ ID NO:8), a truncatedhemoglobin from Chlamydomonas moewusii (SEQ ID NO:9), Tetrahymenapyriformis (SEQ ID NO:10, group I truncated), Paramecium caudatum (SEQID NO:11, group I truncated), a hemoglobin from Aspergillus niger (SEQID NO:12), or a mammalian myoglobin protein such as the Bos taurus (SEQID NO:18) myoglobin, Sus scrofa (SEQ ID NO:19) myoglobin, or Equuscaballus (SEQ ID NO:20) myoglobin, a heme-protein from Nicotianabenthamiana (SEQ ID NO:21), Bacillus subtilis (SEQ ID NO:22),Corynebacterium glutamicum (SEQ ID NO:23), Synechocystis PCC6803 (SEQ IDNO:24), Synechococcus sp. PCC 7335 (SEQ ID NO:25), Nostoc commune (SEQID NO:26), or Bacillus megaterium (SEQ ID NO:27). See FIG. 1.

The percent identity between two amino acid sequences can be determinedas follows. First, the amino acid sequences are aligned using the BLAST2 Sequences (Bl2seq) program from the stand-alone version of BLASTZcontaining BLASTP version 2.0.14. This stand-alone version of BLASTZ canbe obtained from Fish & Richardson's web site (e.g., www.fr.com/blast/)or the U.S. government's National Center for Biotechnology Informationweb site (www.ncbi.nlm.nih.gov). Instructions explaining how to use theBl2seq program can be found in the readme file accompanying BLASTZ.Bl2seq performs a comparison between two amino acid sequences using theBLASTP algorithm. To compare two amino acid sequences, the options ofBl2seq are set as follows: -i is set to a file containing the firstamino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to afile containing the second amino acid sequence to be compared (e.g.,C:\seq2.txt); -p is set to blastp; -o is set to any desired file name(e.g., C:\output.txt); and all other options are left at their defaultsetting. For example, the following command can be used to generate anoutput file containing a comparison between two amino acid sequences:C:\B12seq -i c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. Ifthe two compared sequences share homology, then the designated outputfile will present those regions of homology as aligned sequences. If thetwo compared sequences do not share homology, then the designated outputfile will not present aligned sequences. Similar procedures can befollowing for nucleic acid sequences except that blastn is used.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The percent identity is determined by dividing the number ofmatches by the length of the full-length polypeptide amino acid sequencefollowed by multiplying the resulting value by 100. It is noted that thepercent identity value is rounded to the nearest tenth. For example,78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15,78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It also is notedthat the length value will always be an integer.

It will be appreciated that a number of nucleic acids can encode apolypeptide having a particular amino acid sequence. The degeneracy ofthe genetic code is well known to the art; i.e., for many amino acids,there is more than one nucleotide triplet that serves as the codon forthe amino acid. For example, codons in the coding sequence for a givenenzyme can be modified such that optimal expression in a particularspecies (e.g., bacteria or fungus) is obtained, using appropriate codonbias tables for that species.

Heme-containing proteins can be extracted from the source material(e.g., extracted from animal tissue, or plant, fungal, algal, orbacterial biomass, or from the culture supernatant for secretedproteins) or from a combination of source materials (e.g., multipleplant species). Leghemoglobin is readily available as an unusedby-product of commodity legume crops (e.g., soybean, alfalfa, or pea).The amount of leghemoglobin in the roots of these crops in the UnitedStates exceeds the myoglobin content of all the red meat consumed in theUnited States.

In some embodiments, extracts of heme-containing proteins include one ormore non-heme-containing proteins from the source material (e.g., otheranimal, plant, fungal, algal, or bacterial proteins) or from acombination of source materials (e.g., different animal, plant, fungi,algae, or bacteria).

In some embodiments, heme-containing proteins are isolated and purifiedfrom other components of the source material (e.g., other animal, plant,fungal, algal, or bacterial proteins) using techniques described above.As used herein, the term “isolated and purified” indicates that thepreparation of heme-containing protein is at least 60% pure, e.g.,greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% pure.

Heme-containing proteins also can be recombinantly produced usingpolypeptide expression techniques (e.g., heterologous expressiontechniques using bacterial cells, insect cells, algal cells, fungalcells such as yeast cells, plant cells, or mammalian cells). Forexample, the heme-containing protein can be expressed in E. coli cells.The heme-containing proteins can be tagged with a heterologous aminoacid sequence such as FLAG, polyhistidine (e.g., hexahistidine, HIStag), hemagluttinin (HA), glutathione-S-transferase (GST), ormaltose-binding protein (MBP) to aid in purifying the protein. In someembodiments, a recombinant heme containing protein including a HIS-tagand a protease (e.g., TEV) site to allow cleavage of the HIS-tag, can beexpressed in E. coli and purified using His-tag affinity chromatography(Talon resin, CloneTech). In some cases, standard polypeptide synthesistechniques (e.g., liquid-phase polypeptide synthesis techniques orsolid-phase polypeptide synthesis techniques) can be used to produceheme-containing proteins synthetically. In some cases, cell-freetranslation techniques can be used to produce heme-containing proteinssynthetically.

In some embodiments, the isolated and purified protein is substantiallyin its native fold and water soluble. In some embodiments, the isolatedand purified protein is more than 50, 60, 70, 80, or 90% in its nativefold. In some embodiments, the isolated and purified protein is morethan 50, 60, 70, 80, or 90% water soluble.

The proteins used in the consumable can be altered (e.g., hydrolyzed,cleaved, crosslinked, denatured, polymerized, extruded, electrospun,spray dried or lyophilized, or derivatized or chemically modified). Forexample, the proteins can be modified by covalent attaching sugars,lipids, cofactors, peptides, or other chemical groups includingphosphate, acetate, methyl, and other natural or unnatural molecule. Forexample, the peptide backbones of the proteins can be cleaved byexposure to acid or proteases or other means. For example, the proteinscan be denatured, i.e., their secondary, tertiary, or quaternarystructure can be altered, by exposure to heat or cold, changes in pH,exposure to denaturing agents such as detergents, urea, or otherchaotropic agents, or mechanical stress including shear. The alignmentof proteins in a solution, colloid, or a solid assembly can becontrolled to affect the mechanical properties including tensilestrength, elasticity, deformability, hardness, or hydrophobicity.

Proteins also can be assembled into fibers that can form a matrix for astructure for the compositions. A 3-dimensional matrix of protein fiberscan, for example, contain chemicals that promote formation ofinter-molecular disulfide cross-links (mixed glutathione, dithiothreitol(DTT), beta-mercaptoethanol (BME)). In some embodiments, the chemicalsare proteins (thioredoxin, glutaredoxin). In some embodiments, theproteins are enzymes (disulfide isomerase). In some embodiments, fibersare crosslinked by chemical cross-linkers with two reactive groupsselected from the group consisting of N-hydroxysuccinimide (NHS) esters,imidoesters, aryl fluorides, aldehydes, maleimides, pyridyldithiols,haloacetyls, aryl azides, diazirines, carbodiimides, hydrazides andisocyanates.

In some embodiments, coacervates comprising one or more plant proteinscan be formed and used, for example, as binding agents in meat or otherreplicas. Coacervation is the process during which a homogeneoussolution of charged polymers undergoes a phase separation to result in apolymer-rich dense phase (the ‘coacervate’) and a solvent-rich phase(supernatant). Protein-polysaccharide coacervates have been used in thedevelopment of biomaterials. See, for example, Boral and Bohidar (2010)Journal of Physical Chemistry B. Vol 114 (37): 12027-35; and Liu et al.,(2010) Journal of Agricultural and Food Chemistry, Vol 58:552-556.Formation of such coacervates is driven by associative interactionsbetween oppositely charged polymers. However, as described herein,coacervates can be formed using proteins (e.g., plant proteins compriseone or more pea proteins, chickpea proteins, lentil proteins, lupineproteins, other legume proteins, or mixtures thereof). In general, acoacervate can be formed by acidifying a low ionic strength solution(e.g., a buffered solution at or below 100 mM sodium chloride)comprising one or more isolated and purified plant proteins such as pealegumins or vicilins (e.g., a vicilin fraction comprising convicilins),a combination of both vicilins and legumins, or unfractionated peaproteins to a pH of 3.5 to 5.5. (e.g., pH 4 to 5). Under theseconditions, the proteins separate out of solution and the mixture can becentrifuged to cleanly separate out the coacervate. This coavervate,unlike a precipitate, is a viscous material that can be stretched bypulling and that melts on heating. The process can be carried out in thepresence of oils (up to 70%, e.g., palm or other oil), to form a creamymaterial. By varying the composition of the solution (ratio ofvicilin:legumin, type and amount of oil used), the binding properties ofthe coacervate can be tuned as desired. In some embodiments, one or moregums (e.g., acacia gum or xanthan gum) can be used to form a coacervate.Coacervates can be used as binding agents in beef patty replicas to bindand hold together the adipose-, muscle- and connective tissue replicas.

Binding materials with different adhesive and cooking characteristicscan be prepared by combining wheat gluten (0-20%) and pea proteinfractions (0-50%) in the presence of a plasticizer such as glycerol(0-30%) or polyethylene glycol. Leghemoglobin or other heme-containingprotein can be added to the mixture if necessary. Upon mixing to removeany clumps, the material may be incorporated into beef patty replicas.

In some embodiments, proteins can be subjected to freeze alignment totexturize the proteins without extrusion. The method involves slowfreezing of protein comprising materials to allow for formation of icecrystals. When cooled from one side, ice crystals form preferentially ina direction perpendicular to the cooled side. After freezing, the icecan be removed from the material in a freeze-dryer, leaving behindmaterial with several layers. The structure can then be stabilized byheating under pressurized, moist conditions to produce a material thatcan be used in meat replicas. Freeze-alignment of soy proteins has beendescribed by Lugay and Kim (1981) (see Freeze alignment: A novel methodfor protein texturization. Page 177-187, Chapter 8 in: D. W. Stanley, E.D. Murray and D. H. Lees eds. 1981. Utilization of Protein Resources.Westport, Conn.: Food & Nutrition Press, Inc). The freeze-alignedproteins can be subjected to further processing (by soaking in solutionscomprising beef flavors and/or leghemoglobin) and used in combinationwith adipose- and connective-tissue replicas to form beef replicas. Thereplicas may also be used as structures around which cold-set gels(comprising, for example, pea proteins and myoglobin) or crosslinkedgels (comprising, for example, pea proteins and leghemoglobin) can beformed prior to their combination with adipose- and connective-tissues.

C. Lipids

Consumables described herein can include a lipid component. Lipids canbe isolated and/or purified and can be in the form of triglycerides,monoglycerides, diglycerides, free fatty acids, sphingosides,glycolipids, phospholipids, or oils, or assemblies of such lipids (e.g.,membranes, lecithin, lysolecithin, or fat droplets containing a smallamount of lipid in a bulk water phase). In some embodiments, the lipidsources are oils obtained from non-animal sources (e.g., oils obtainedfrom plants, algae, fungi such as yeast or filamentous fungi, seaweed,bacteria, or Archae), including genetically engineered bacteria, algae,archaea or fungi. Non-limiting examples of plant oils include corn oil,olive oil, soy oil, peanut oil, walnut oil, almond oil, sesame oil,cottonseed oil, rapeseed oil, canola oil, safflower oil, sunflower oil,flax seed oil, palm oil, palm kernel oil, coconut oil, babassu oil, sheabutter, mango butter, cocoa butter, wheat germ oil, or rice bran oil; ormargarine. The oils can be hydrogenated (e.g., a hydrogenated vegetableoil) or non-hydrogenated.

In some embodiments, the lipid can be triglycerides, monoglycerides,diglycerides, free fatty acids, sphingosides, glycolipids, lecithin,lysolecithin, phospholipids such as phosphatidic acids, lysophosphatidicacids, phosphatidyl cholines, phosphatidyl inositols, phosphatidylethanolamines, or phosphatidyl serines; sphingolipids such assphingomyelins or ceramides; sterols such as stigmasterol, sitosterol,campesterol, brassicasterol, sitostanol, campestanol, ergosterol,zymosterol, fecosterol, dinosterol, lanosterol, cholesterol, orepisterol; lipid amides, such as N-palmitoyl proline, N-stearoylglycine, N-palmitoyl glycine, N-arachidonoyl glycine, N-palmitoyltaurine, N-arachidonoyl histidine, or anandamide; free fatty acids suchas palmitoleic acid, palmitic acid, myristic acid, lauric acid,myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonicacid, undecanoic acid, linoleic acid (C18:2), eicosanoic acid (C22:0),arachidonic acid (C20:4), eicosapentanoic acid (C20:5), docosapentaenoicacid (C22:5), docosahexanoic acid (C22:6), erucic acid (C22:1),conjugated linoleic acid, linolenic acid (C18:3), oleic acid (C18:1),elaidic acid (trans isomer of oleic acid), trans-vaccenic acid (C18:1trans 11), or conjugated oleic acid; or esters of such fatty acids,including monoacylglyceride esters, diacylglyceride esters, andtriacylglyceride esters of such fatty acids.

The lipids can comprise phospholipids, lipid amides, sterols or neutrallipids. The phospholipids can comprise a plurality of amphipathicmolecules comprising fatty acids (e.g., see above), glycerol and polargroups. In some embodiments, the polar groups are, for example, choline,ethanolamine, serine, phosphate, glycerol-3-phosphate, inositol orinositol phosphates. In some embodiments, the lipids are, for example,sphingolipids, ceramides, sphingomyelins, cerebrosides, gangliosides,ether lipids, plasmalogens or pegylated lipids.

In some embodiments, the lipids used in the consumable are the creamfraction created from seeds, nuts, and legumes, including but notlimited to sunflower seeds, safflower seeds, sesame seeds, rape seeds,almonds, macadamia, grapefruit, lemon, orange, watermelon, pumpkin,cocoa, coconut, mango, butternut squash, cashews, brazilnuts, chestnuts,hazelnuts, peanuts, pecans, walnuts and pistachios. As used herein, theterm “cream fraction” can refer to an isolated emulsion comprisinglipids, proteins and water.

To obtain a cream fraction from seeds, nuts, or legumes, one or more ofthe following steps can be performed. Seeds, nuts or legumes can beblended from 1 minute up to 30 minutes. For example, the seeds, nuts, orlegumes can be blended by increasing the speed gradually to maximumspeed over 4 minutes, then blending at maximum speed for 1 minute. Theseeds, nuts or legumes can be blended in water or solutions that containall or some of the following: EDTA (0-0.1M), NaCl (0-1M), KCl (0-1M),NaSO₄ (0-0.2M), potassium phosphate (0-1M), sodium citrate (0-1M),sodium carbonate (0-1M), and/or sucrose (0-50%), from pH of 3 to 11 toobtain a slurry. The slurry can be heated to 20° C. to 50° C. andcentrifuged to obtain the cream fraction (the top layer, also referredto as the “cream”). Further purification of the cream fraction may beachieved by washing the cream fraction with 0.1M to 2M urea solutionbefore re-isolating the cream fraction by centrifugation. The residualliquid (referred to as the “skim” layer) that is a solution comprisingproteins in water can also be used.

The “cream” can be used as is, or subjected to further purificationsteps. For example, washing and heating can remove color and flavormolecules (e.g., unwanted molecules), or unwanted grainy particles toimprove the mouth feel and creaminess. In particular, washing with ahigh pH buffer (pH>9) can remove bitter tasting compounds and improvemouth feel, washing with urea can remove storage proteins, washing belowpH 9, followed by washing with a pH above pH 9 can remove unwanted colormolecules, and/or washing with salts can decrease taste compounds.Heating can increase the removal of grainy particles, color and flavorcompounds. For example, the cream fraction can be heated from 0-24hours, at temperatures ranging from 25° C. to 80° C. In someembodiments, the resulting creamy fraction comprises seed storageproteins. In some embodiments, the seed storage proteins aresubstantially removed from the resulting creamy fraction.

D. Fiber

Fiber can be isolated and/or purified for inclusion in the consumablesdescribed herein. Fiber can refer to non-starch polysaccharides such asarabinoxylans, cellulose, and other plant components such as resistantstarch, resistant dextrins, inulin, lignin, waxes, chitins, pectins,beta-glucans, and oligosaccharides from any plant source.

Fibers can refer to extruded and solution spun proteins as describedherein.

E. Sugars

In some embodiments, the consumable also can comprise sugars. Forexample the consumable can comprise: monosaccharides, including but notlimited to glucose (dextrose), fructose (levulose), galactose, mannose,arabinose, xylose (D- or L-xylose), and ribose, disaccharides includingbut not limited to sucrose, lactose, melibiose, trehalose, cellobiose,or maltose, sugar alcohols such as arabitol, mannitol, dulcitol, orsorbitol, sugar acids such as galacturonate, glucuronate, or gluconate,oligosaccharides and polysaccharides such as glucans, starches such ascorn starch, potato starch, pectins such as apple pectin or orangepectin, raffinose, stachyose, or dextrans; plant cell wall degradationproducts such as salicin, and/or sugar derivatives such asN-acetylglucosamine.

F. Gel Formation

The components of the composition can be formed into a gel. In someembodiments, gels comprise protein, where the protein is derived fromnon-animal source (e.g., a plant source or other non-animal source suchas a genetically modified yeast or bacteria). Gels can be formed using avariety of methods. The protein concentration, enzyme concentration, pH,and/or process temperature will affect the rate of gel formation andquality of the final tissue replica.

Gels can be stabilized entirely by physical cross-links between thecomponents. In some embodiments, gels can be produced by heat/coolcycles, in which case the gel is stabilized by physical interactions(entanglements, hydrophobic interactions) between protein molecules. Forexample, a gel can be formed by heating a protein solution to atemperature of at least 40° C., 45° C., 50° C., 60° C., 70° C., 80° C.,90° C., or 100° C. and then cooling to room temperature, or to atemperature below 40° C.

In some embodiments, a gel can be formed by subjecting the compositioncontaining the protein and any other components (e.g., a lipid) to highpressure processing.

In some embodiments, gels can be produced by adjusting the pH of thesolution. For example, the pH of a concentrated protein solution can beadjusted to near the isolectric pH of the main protein component byadding hydrochloric acid or other acid, or sodium hydroxide or otherbase.

In some embodiments, gels can be produced by soaking protein powders insolutions. For example, protein powder can be soaked with at least 1%,5%, 10%, 20% (wt/v) or more of a concentrated sodium hydroxide solution.In other examples, protein powder can be soaked in mixed water/ethanolsolutions.

In some embodiments, a cold set gel is formed to avoid denaturing or thebreakdown of any heat-labile components (e.g., oxidizing the iron in aheme moiety or generating undesirable flavors). See, Ju and Kilara A.(1998) J. Food Science, Vol 63(2): 288-292; and Maltais et al., (2005)J. Food Science, Vol 70 (1): C67-C73) for general methodologies forforming cold set gels. In general, cold set gels are formed by firstheat denaturing a protein solution below its minimum gellingconcentration (dependent on pH and type of protein, typically <8% (w/v)at pH 6-9 for globular plant proteins such as pea proteins). The proteinsolution can heated to a temperature above the denaturation temperatureof the protein under conditions where it does not precipitate out ofsolution (e.g., 0-500 mM sodium chloride, pH 6-9). The solution can becooled back to room temperature or below, and any heat-labile components(e.g., heme-containing proteins and/or oils) can be mixed in when thesolution is sufficiently cool, but before gelling. Gelation can beinduced by adding sodium chloride or calcium chloride (e.g., 5 to 100mM), and the solution can be incubated at or below room temperature toallow for gel formation (typically minutes-hours). The resulting gel canbe used as-is in meat replicas or processed further (e.g., stabilized)before incorporation in meat replicas.

In some embodiments, gels can comprise or be produced (e.g., stabilizedby) at least in part by a cross-linking enzyme. The cross-linking enzymecan be, for example, a transglutaminase, a tyrosinase, a lipoxygenase, aprotein disulfide reductase, a protein disulfide isomerase, a sulfhydryloxidase, a peroxidase, a hexose oxidase, a lysyl oxidase, or an amineoxidase.

In some cases, gels can comprise chemicals that promote formation ofinter-molecular disulfide cross-links between the proteins. In someembodiments, the chemicals are proteins (e.g., thioredoxin,glutaredoxin). In some embodiments, the proteins are enzymes (disulfideisomerase).

Gels can be stabilized by chemical crosslinking by chemicalcross-linkers with two reactive groups selected from the groupconsisting of N-hydroxysuccinimide (NHS) esters, imidoesters, arylfluorides, aldehydes, maleimides, pyridyldithiols, haloacetyls, arylazides, diazirines, carbodiimides, hydrazides and isocyanates.

In some embodiments, gels can be stabilized by the addition of starchesand gums.

In some embodiments, more than one of these approaches are used incombination. For example, a transglutaminase cross-linked gel can befurther stabilized by a heat/cool treatment.

G. Muscle Replicas

A large number of meat products comprise a high proportion of skeletalmuscle. Accordingly, the present invention provides a composition, whichcan be derived from non-animal sources which replicates or approximateskey features of animal skeletal muscle. A composition derived fromnon-animal sources, which replicates or approximates animal skeletalmuscle can be used as a component of a consumable, for example, a meatreplica. Such a composition will be labeled herein as “muscle replica.”In some embodiments, the muscle replica and/or meat substitute productcomprising the muscle replica are partially derived from animal sources.In some embodiments, the muscle replica and/or meat substitute productcomprising the muscle replica are entirely derived from non-animalsources.

The muscle tissue replica can comprise a protein content, wherein theprotein content comprises one or more isolated and purified proteins,wherein the muscle tissue replica approximates the taste, texture, orcolor of an equivalent muscle tissue derived from an animal source.

Many meat products comprise a high proportion of striated skeletalmuscle in which individual muscle fibers are organized mainly in ananisotropic fashion. Accordingly, in some embodiments, the musclereplica comprises fibers that are to some extent organizedanisotropically. The fibers can comprise a protein component. In someembodiments, the fibers comprise about 1% (wt/wt), about 2%, about 5%,about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, about 95%, about 99% (wt/wt) ormore of a protein component.

The connective tissue component of skeletal muscle substantiallycontributes to texture, mouth feel and cooking behavior of meatproducts. Connective tissue is composed of protein (collagen, elastin)fibers in the range of 0.1-20 microns. In some embodiments, a mixture offibers of diameters of <1-10 microns, and 10-300 microns is produced toreplicate the fiber composition of animal connective tissue. In someembodiments, the 3-dimensional matrix of fibers is stabilized by proteincrosslinks to replicate the tensile strength of animal connectivetissue. In some embodiments, the 3-dimensional matrix of fibers containsan isolated, purified cross-linking enzyme. The cross-linking enzyme canbe, for example, a transglutaminase, a tyrosinase, a lipoxygenase, aprotein disulfide reductase, a protein disulfide isomerase, a sulfhydryloxidase, a peroxidase, a hexose oxidase, a lysyl oxidase, or an amineoxidase.

Some proteins (e.g., 8S globulin from Moong bean seeds, or the albuminor globulin fraction of pea seeds) have favorable properties forconstructing meat replicas because of their ability to form gels withtextures similar to animal muscle or adipose tissue. See also theproteins identified in Section III A and B. The proteins may beartificially designed to emulate physical properties of animal muscletissue.

In some embodiments, one or more isolated and purified proteins accountsfor about 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99% or more of the protein component by weight of themeat replica. In some embodiments, one or more isolated and purifiedproteins accounts for about 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the protein content of aconsumable.

Skeletal muscles of animals such as beef cattle typically containsubstantial quantities of glycogen, which can comprise on the order of1% of the mass of the muscle tissue at the time of slaughter. Afterslaughter, a fraction of this glycogen continues to be metabolized,yielding products including lactic acid, which contributes to loweringthe pH of the muscle tissue, a desirable quality in meat. Glycogen is abranched polymer of glucose linked together by alpha (1->4) glycosidicbonds in linear chains, with branch points comprising alpha (1->6)glycosidic bonds. Starches from plants, particularly amylopectins arealso branched polymers of glucose linked together by alpha (1->4)glycosidic bonds in linear chains, with branch points comprising alpha(1->6) glycosidic bonds and can therefore be used as an analog ofglycogen in constructing meat replicas. Thus, in some embodiments, themuscle or meat replica includes a starch or pectin.

Additional components of animal muscle tissue include sodium, potassium,calcium, magnesium, and other metal ions, lactic acid and other organicacids, free amino acids, peptides, nucleotides, and sulfur compounds.Thus, in some embodiments, a muscle replica can include sodium,potassium, calcium, magnesium, other metal ions such as iron, zinc,copper, nickel, lithium, or selenium, lactic acid, and other organicacids such as fatty acids, free amino acids, peptides, nucleotides andsulfur compounds glutathione, beta mercaptoethanol, or dithiothreitol.In some embodiments, the concentration of sodium, potassium, calcium,magnesium, other metal ions, lactic acid, other organic acids, freeamino acids, peptides, nucleotides and/or sulfur compounds in the musclereplica or consumable are within 10% of the concentrations found in amuscle or meat being replicated.

The invention also provides methods for making a muscle replica. In someembodiments, the method includes forming the composition into asymmetricfibers prior to incorporation into the consumable. In some embodiments,these fibers replicate muscle fibers. In some embodiments the fibers arespun fibers. In other embodiments the fibers are extruded fibers.Accordingly, the present invention provides for methods for producingasymmetric or spun protein fibers. In some embodiments, the fibers areformed by extrusion of the protein component through an extruder.Methods of extrusion are well known in the art, and are described, forexample, in U.S. Pat. Nos. 6,379,738, 3,693,533, and U.S. PatentPublication No. 20120093994, which are herein incorporated by reference.These methods can be applied to making the compositions provided herein.

Extrusion can be conducted using, for example, a Leistritz Nano-16twin-screw co-rotating extruder (American Leistritz Extruder Corp. USA,Sommerville, N.J.). Active cooling of the barrel section can be used tolimit denaturation of proteins. Active cooling of the die section can beused to limit expansion of the extruded product and excessive moistureloss. Protein feed and liquid are added separately: protein is fed by avolumetric plunger feeder or a continuous auger-type feeder, and liquidcan be added into the barrel through a high pressure liquid injectionsystem. Die nozzles with various inner diameters and channel length canbe used for precise control of extrudiate pressure, cooling rate andproduct expansion. In some examples, extrusion parameters were: screwspeed 100-200 rpm, die diameter 3 mm, die length 15 cm, producttemperature at the end of the die of 50° C., feed rate of 2 g/min, andwater-flow rate of 3 g/min. Product temperature at the die duringextrusion is measured by a thermocouple.

Spun fibers can be produced by preparing a high viscosity protein “dope”by adding sodium hydroxide to concentrated protein solutions or toprecipitated proteins, and forcing the solution with a plunger-typedevice (in some examples, a syringe with a syringe pump) through a smallsteel capillary (in some examples, 27 gauge hypodermic needle) into acoagulating bath. In some examples, the bath is filled with aconcentrated acid solution (e.g. 3 M hydrochloric acid). In someexamples, the bath is filled with a buffer solution at a pHapproximately equal to the isoionic point of the protein. Coagulatingprotein solution jet forms a fiber that collects at the bottom of thebath.

Bundles of spun fibers can be produced by forcing protein “dope” throughspinnerets with many small holes. In some examples, spinnerets arestainless steel plates with approximately 25,000 holes per cm², withdiameter of each hole approximately 200 microns. In some embodimentsmuscle tissue replica is produced by immersing the 3-dimensional matrixof fibers (connective tissue replica) in solutions of proteins andcreating protein gels incorporating 3-dimensional matrix of fibers.

H. Fat Replicas

Animal fat is important for the experience of eating cooked meat and areimportant for some of the nutritional value of meat. Accordingly, thepresent invention provides compositions derived from non-animal sources,which recapitulate key features of animal fat, including the textureand/or flavor, by using components that mimic the chemical compositionand physical properties of, for example, ground beef. In another aspect,the present invention provides a meat substitute product that comprisesa composition derived from non-animal sources, which recapitulatesanimal fat. Such a composition will be labeled herein as an “adiposereplica” or a “fat replica.” In some embodiments, the adipose replicaand/or meat substitute product comprising the adipose replica arepartially derived from animal sources. The consumable can also includeadipose replicas that recapitulate key features of non-animal fats,including texture, flavor, firmness, percent fat release, and/ortemperature of fat release. The fat content of the consumable can be atleast 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,70%, 80%, 90%, or 95% fat.

Ground beef is typically prepared by mixing lean beef with adipose (fat)that is cut from steaks, with the adipose tissue added to 16-30% (Cox1993). Without adipose, meat passed through a grinder is tough, crumbly,and dries out quickly. Fat is added to lean beef so that fat releasedduring cooking provides a liquid surface to aid in cooking, and togenerate key beef flavors, which largely are products of the fattyacids. Engineering an adipose tissue replica that plays the same keyroles in texture and flavor of the plant-based ground beef is animportant driver for texture and flavor.

The adipose tissue replicas described here have a great health benefitover the beef adipose tissue as the fatty acid composition can becontrolled such that the amount of saturated fats can be decreased.Additionally, the plant-based adipose replicas are cholesterol free. Theplant-based adipose replicas can contain a lower percent total fat, andstill have the same amount of fat being released or retained for thedesired cooking properties, flavor, and texture.

As described herein, adipose replicas comprising emulsions ofplant-derived lipids and one or more isolated and purified proteins canbe produced in which the composition (e.g., fatty acid composition),cooking characteristics (e.g., fat release temperature or percent fatrelease), and physical properties (e.g., firmness) can be controlled,allowing the plant based composition to mimic animal based adipose. Theadipose tissue replica includes (1) a vegetable oil containingtriacylglycerides of fatty acids; (2) one or more isolated and purifiedproteins from non-animal sources (e.g., a plant protein); and (3) aphospholipid such as lecithin. The proteins can be plant or microbialproteins as described above (e.g., RuBisCo, an oleosin, an albumin, aglobulin, or other seed storage protein). See, also the proteinsdescribed in Sections III A and B. The vegetable oils can be any of theoils described herein. See, e.g., Section III C.

The fat replica can be a gelled emulsion. In some embodiments, the gelis a soft, elastic gel comprising proteins and optionally carbohydrates.The gelled emulsion can comprise a protein solution comprising multipleproteins, e.g., 1-5 or 1-3 isolated and purified proteins, wherein theprotein solution accounts for 1-30% of the volume of the emulsion. Thegelled emulsion can comprise a fat droplet, wherein the fat dropletaccounts for 70-99% of the volume of the emulsion. The gelled emulsioncan comprise an isolated, purified cross-linking enzyme, wherein thecross-linking enzyme accounts for 0.0005% to 0.5% of the emulsion weightby volume, 0.5-2.5% of the emulsion weight by volume, or 0.001% or lowerof the emulsion weight by volume. The emulsion of fat droplets in theprotein solution can be stabilized by forming the emulsion into a gel bythe cross-linking enzyme, e.g., a transglutaminase, by gelling proteinsvia heating and cooling protein solutions, by forming a cold-set gel, byformation of a coacervate, or by combinations of these techniques asdescribed for coacervates in section C and gel formation in section F.

In some embodiments, the fat replica comprises cross-linking enzymesthat catalyze reactions leading to covalent crosslinks between proteins.Cross-linking enzymes can be used to create or stabilize the desiredstructure and texture of the adipose tissue replica, to mimic thedesired texture of an equivalent desired animal fat. In someembodiments, the cross-linking enzymes are isolated and purified from anon-animal source, examples and embodiments of which are describedherein. In some embodiments, the fat replica comprises at least 0.0001%,at least 0.001%, at least 0.01%, at least 0.1%, or at least 1% (wt/vol)of a cross-linking enzyme. The cross-linking enzyme can be selectedfrom, for example, transglutaminases, tyrosinases, lipoxygenases,protein disulfide reductases, protein disulfide isomerases, sulfhydryloxidases, peroxidases, hexose oxidases, lysyl oxidases, and amineoxidases. In some embodiments, the cross-linking enzyme istransglutaminase, a lysyl oxidase (e.g., a Pichia pastoris lysyloxidase), or other amine oxidase.

The fat replica can comprise a gel with droplets of fat suspendedtherein. The fat droplets used in some embodiments of the presentinvention can be from a variety of sources. In some embodiments, thesources are non-animal sources (e.g., plant sources). See, e.g., theexamples provided in Section III C. In some embodiments, the fatdroplets are derived from animal products (e.g., butter, cream, lard,and/or suet). In some embodiments, fat droplets are derived from pulp orseed oil. In other embodiments, the source may be algae, yeasts,oleaginous yeasts such as Yarrowia lipolytica, or mold. For instance, inone embodiment, triglycerides derived from Mortierella isabellina can beused. In some embodiments, the fat droplets contain synthetic orpartially synthetic lipids

In some embodiments, the fat droplets are stabilized by addition ofsurfactants, including but not limited to phospholipids, lecithins, andlipid membranes. The lipid membranes may be derived from algae, fungi orplants. In some embodiments the surfactants comprise less than 5% of thefat replica. The fat droplets can in some examples range from 100 nm to150 μm in diameter. The diameter of these stabilized droplets may beobtained by homogenization, high-pressure homogenization, extrusion orsonication.

In some embodiments, plant oils are modified to resemble animal fats.The plant oils can be modified with flavoring or other agents such asheme proteins, amino acids, organic acids, lipids, alcohols, aldehydes,ketones, lactones, furans, sugars, or other flavor precursor, torecapitulate the taste and smell of meat during and after cooking.Accordingly, some aspects of the invention involve methods for testingthe qualitative similarity between the cooking properties of animal fatand the cooking properties of plant oils in the consumable.

In some embodiments, additional polysaccharides can be added to a fatreplica, including flax seed polysaccharides and xanthan gum.

The creation of a plant-based adipose replica requires stabilization ofthe oil in water emulsions. Typically animal adipose tissue contains˜95% fat, and is stabilized by the phospholipid bilayer and associatedproteins. Adipose replicas described herein can be created with up to95% fat in some instances, with 80% fat under many conditions, or withlower amounts of fat (e.g., 50% or less) while mimicking the propertiesof animal fat. Achieving a high percent fat is controlled by thestabilization of the emulsion.

The composition (e.g., fatty acid composition), cooking characteristics(e.g., fat release temperature or percent fat release), and physicalproperties (e.g., firmness) can be manipulated by controlling the typeand amount of fat, the amount of protein, the type and amount oflecithin, the presence of additives, and the method of gelling.

In some embodiments, the protein component comprises about 0.1%, 0.5%,1%, 2%, 5%, 10%, 15%, or 20%, 25%, or more of the fat replica by dryweight or total weight. In some embodiments, the protein componentcomprises about 0.1-5% or about 0.5-10% or more of the fat replica bydry weight or total weight. In some embodiments, the protein componentis 0.5 to 3.5% or 1 to 3% of the fat replica by dry weight or totalweight. In some embodiments, the protein component comprises a solutioncontaining one or more isolated, purified proteins. The type of proteincan affect the stability of the emulsion, RuBisCo and pea albumins allowfor fat replicas to made a greater than 90% fat. Addition ofpolysaccharides including flax seed and xanthan gum aid in emulsifyingthe mixture, allowing for an increase in fat content.

The type and amount of fat can be controlled by choosing the source ofthe fat and its lipid composition. In general, oils with higher amountsof saturated fatty acids are better able to be emulsified at lowerprotein concentrations, while oils with more unsaturated fatty acidsrequire higher protein concentrations to be emulsified. Protein isrequired to stabilize the emulsion, and an increase in protein contentincreases the stability. If the amount of protein added is too little toemulsify the amount of fat, the mixture will separate into layers.

Lecithin also is a modulator of emulsion, and can either stabilize ordisrupt the emulation depending on the amount of protein present andtype of oil used. For example, lecithin may disrupt the protein/fatmatrix to make a less stable emulsion, but can be added at low levels tomodulate other physical properties. Emulsions made from oils with higheramounts of unsaturated fats can be de-stabilized by a high amount oflecithin (1%), such that the emulsion does not solidify. Emulsions madefrom oils with higher amounts of saturated fats can solidify at highamounts of lecithin (1%), but are very soft.

As described herein adipose replicas can be prepared that can range fromvery soft to very firm. The composition and amount of the fat controlsthe firmness of the replica. Firmer oils, which contain morelong-chained saturated fats, make firmer gels. Oils that produce softergels typically contain more unsaturated fatty acids or short-chainedsaturated fatty acids. In general, the firmness of the gel increases astotal percent fat increases, as long as the emulsion is held and doesnot separate. The amount of protein also contributes to the firmness ofthe replica. In general, an increase in protein concentration increasesreplica firmness. The amount of lecithin is a modulator of replicafirmness. Higher amounts of lecithin (1%) are much softer than loweramounts of lecithin (0.05%) when gels are formed with high percentprotein (3%). When protein is decreased (1.8%), all gels are softer, andthere is little difference in firmness between low level of lecithin(0.05%) and high (1%) if emulsion is held.

Addition of polysaccharides to replicas including but not limited toxanthan gum and flax seed paste can increase the firmness ofadipose-replica gels.

When an adipose replica is cooked, fat leaks from the structured replicaas it is cooked. Often there is fat that remains in the cooked product;it is important to achieve the balance between fat released to aid incooking and fat retained for texture and taste. The percent fat released(per total fat) can be determined by measuring the amount of fatreleased upon cooking to completion. Percent fat released is reported asthe weight of fat released per the total fat of the replica. Forexample, the percent fat release of an adipose tissue replica describedherein can be 0 to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%,50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% uponcooking. Adipose replicas typically release 0-90% fat under standardcooking conditions. In comparison, beef adipose tissue typicallyreleases 40-55% fat under equivalent conditions.

While vegetable oils have set melting temperatures, the range oftemperatures over which adipose replicas can be made to release fat iswide. Fat release temperature is the temperature at which fat is visiblyreleased from the replica at the cooking surface. As described herein,the fat release temperature of the adipose replica can be tailored basedon the type and amount of fat, the amount of protein, the type andamount of lecithin, the presence of additives, the method ofemulsification, and the method of gelling. The resulting adiposereplicas can have a fat release temperature of between 23° C. to 33° C.,34° C. to 44° C., 45° C. to 55° C., 56° C. to 66° C., 67° C. to 77° C.,78° C. to 88° C., 89° C. to 99° C., 100° C. to 110° C., 111° C. to 121°C., 122° C. to 132° C., 133° C. to 143° C., 144° C. to 154° C., 155° C.to 165° C., 166° C. to 167° C., 168° C. to 169° C., 170° C. to 180° C.,181° C. to 191° C., 192° C. to 202° C., 203° C. to 213° C., 214° C. to224° C., 225° C. to 235° C., 236° C. to 246° C., 247° C. to 257° C.,258° C. to 268° C., 269° C. to 279° C., 280° C. to 290° C., or 291° C.to 301° C. Beef fat was measured to release fat at 100-150° C.

Emulsification also is a factor in controlling the temperature of fatrelease: Once fats are incorporated into a replica with protein, orprotein and lecithin, the temperature at which the fat is releasedincreases significantly above the temperature at which the fat alonemelts.

Fatty acid composition also is a factor in the fat release temperatureand percent fat release. Vegetable oils that contain a higher proportionof unsaturated fatty acids have low melting temperatures and many areliquid at room temperature. Vegetable oils that contain a higherproportion of saturated fatty acids have a higher melting temperature,and are solid at room temperature. Replicas with a greater amount ofunsaturated fats have a higher temperature of fat leakage than the samereplica made with more saturated fatty acids. Gels made from 75% oilswith higher amounts of unsaturated fatty acids, a high protein content(3%), and minimal lecithin content (0.05%), where the mixture wasemulsified by a hand-held homogenizer and gelled using the heat-coolmethod, can be heated to 200° C. with little or no fat release. Replicascontaining oils with more long-chained saturated fats typically havemore fat release at high protein content, but release less percent totalfat compared to replicas containing oils with more short chained fatsand at low percent protein. Gels made from oils with a higher proportionof short-chained saturated fatty acids, high protein content (3%), andminimal lecithin content (0.05%), can be heated to 200° C. with littlefat release.

The percent of fat release as an adipose replica is cooked is also is afunction of the amount of protein and the amount of lecithin. Typically,the adipose replica contains 1-3% protein by mass. Increasing proteincontent leads to increasing temperatures of fat release, and reduces thefraction of fat released. Increasing the lecithin content to 1% candecrease the fat release temperature to 60-115° C., and increase thefraction of fat released (e.g., 25-30%). The source or composition ofthe lecithin used can modulate the amount of fat release and thetemperature threshold for fat release. Without being bound to aparticular mechanism, it is thought that lecithin destabilizes theemulsion by disrupting the protein-protein interactions. In oneembodiment, at a high protein concentration of 3%, increasing thelecithin content to 1% decreased the fat release temperature to 55-60°C., and increased the percent fat leaked to 60-65%.

The method of making the emulsion also is a factor in determining theamount of fat release. Emulsification forms a homogenous mixture of thefat held in a matrix of proteins and lecithin. Methods of emulsificationcan include high-pressure homogenization, sonication, or handhomogenization. The alternative methods result in characteristicdifferences in the size of the oil droplets in the emulsion, whichinfluences the stability of the resulting emulsions and the maximum fatconcentration at which stable emulsions can be formed.

The method of gelling the replica also is a factor in determining theamount of fat release. While adipose replicas can be formed withoutforming gels, gelation results in a firmer and more stable emulsion.Methods of gelling are described above, and can include, for example,the addition of a crosslinking enzyme such as a transglutaminase (TG),or subjecting the emulsion to a heat/cool cycle. For example, eithertreatment with TG or the heat/cool method can convert an emulsion, asdescribed above, into a gel. Furthermore, gelled emulsions formed bycrosslinking catalyzed by TG typically release fat at a temperaturehigher than that at which emulsions gelled by the heat/cool technique doso. Gels formed by crosslinking with a TG also typically release lessfat than do gels formed by the heat-cool technique.

In some embodiments, a fat replica can be made with a protein content<1.5% and a minimal lecithin content (0.05%) and have a fat releasetemperature of 45-65° C., and a high amount of fat released (e.g.,70-90%). These gels are at the higher end of percent fat released.

In some embodiments, a fat replica can be made with a lower proteincontent (<1.5%) and a high lecithin (>1%), and have a lower fat releasetemperature (e.g., 30-50° C., e.g., 30 to 45° C.), and with anintermediate percent fat leaked (45-65%). Thus, in gels formed from oilswith short-chained fatty acids or long-chained fatty acids at lowprotein concentrations, lecithin may play a role in stabilizing theemulsion.

In some embodiments, >2% rubisco or pea albumin can be used to produceadipose replicas with greater than 70% fat. In some embodiments, gelsformed with >3% isolated and purified protein can result in adiposereplicas with greater than 70% fat.

In some embodiments, adipose replicas made from oils with a higherproportion of long-chained saturated fatty acids, a protein content of3%, and a minimal lecithin content (0.05%), can release fat at atemperature similar to that at which beef fat does so (50-100° C.), andcan release a low to intermediate level of fat (15-45%).

In some embodiments, an adipose replica with a higher proteinconcentration (>3%), and a lecithin content >1% can have a fat releasetemperature of 50-70° C., and a higher amount of fat release (50-80%).At high protein and low lecithin concentration, gels with highersaturated fatty acids typically leak about 10% more fat than docorresponding gels formed with unsaturated fats.

In some embodiments, protease treatment of the protein constituentsbefore gel formation can lead to an increase in fat release.

In some embodiments, a adipose tissue replica matrix stabilized bycrosslinking enzymes releases more fat than a adipose tissue replicamatrix stabilized by heat/cool protein denaturation. In one embodiment,an adipose tissue matrix comprised of moong bean 8S protein and canolaoil, or an equal mixture of coconut, cocoa, olive, and palm oils,retains more mass when formed upon heat/cool denaturation than whenformed by cross-linking with an enzyme. In one embodiment, an adiposetissue matrix formed by heat/cool denaturation of a preformedprotein-oil emulsion containing Rubisco and cocoa butter, has a highermelting temperature than an adipose replica of similar compositionstabilized by a cross-linking enzyme.

In some embodiments, adipose tissue replicas constructed from 1.4% wt/vmoong bean 8S protein with 90% v/v canola oil and 0.45% wt/v soybeanlecithin, can be homogenized in the presence of variable concentrationsof sunflower oleosins. Concentration of oleosins can be varied from 1:10to 1:10⁶ molar ratio of oleosin:triglyceride. An increase in massretention after cooking is observed as the concentration of oleosins inthe adipose tissue replica increase.

Firmness of an adipose tissue replica constructed as stabilizedprotein-fat emulsion can be modified by varying the concentration of theprotein within adiopose tissue replica matrix. For example, a series ofadipose tissue replicas formed with varying concentrations of Rubiscowith 70-80% v/v sunflower oil, varied in firmness. Adipose tissuereplicas with 0% and 0.18% (wt/vol) Rubisco were very soft, whereasreplicas formed with 1.6% (wt/vol) Rubisco were soft, and replicasformed with 1.9% (wt/vol) Rubisco were medium in firmness.

In one embodiment, the firmness of adipose replica formed by stabilizingprotein oil emulsion can be modified by varying amount of protein in theadipose replica. In one embodiment, adipose tissue replicas made fromRubisco and 70% sunflower oil are softer at lower concentrations, suchas 1%, of RuBisCo than at higher concentrations of Rubisco, such as 3%,in adipose tissue replicas.

In another aspect, the invention provides methods for making a fatreplica. The fat can be isolated and homogenized. For example an organicsolvent mixture can be used to help solubilize a lipid in a gel and thenremoved to provide the final gel. At this point, the lipid can befrozen, lyophilized, or stored. So in one aspect, the invention providesfor a method for isolating and storing a lipid, which has been selectedto have characteristics similar to animal fat. The lipid film or cakecan then be hydrated. The hydration can utilize agitation or temperaturechanges. The hydration can occur in a precursor solution to a gel. Afterhydration the lipid suspension can be sonicated, homogenized,high-pressure homogenized or extruded to further alter the properties ofthe lipid in the solution.

In some embodiments, the fat replica is assembled to approximate theorganization of adipose tissue in meat. In some embodiments some or allof the components of the fat replica are suspended in a gel (e.g.,proteinaceous gel). In other embodiments, the gel can be a hydrogel, anorganogel, or a xerogel. In some embodiments, the gel can be thickenedto a desired consistency using an agent based on polysaccharides orproteins. For example fecula, arrowroot, cornstarch, katakuri starch,potato starch, sago, tapioca, alginin, guar gum, locust bean gum,xanthan gum, collagen, egg whites, furcellaran, gelatin, agar,carrageenan, cellulose, methylcellulose, hydroxymethylcellulose, acadiagum, konjac, starch, pectin, amylopectin or proteins derived fromlegumes, grains, nuts, other seeds, leaves, algae, bacteria, of fungican be used alone or in combination to thicken the gel, forming anarchitecture or structure for the consumable.

In some embodiments, the tensile strength of the fat replica mimics thetensile strength of adipose tissue. The tensile strength of the gelledemulsions can be increased by incorporation of fibers. The fibers may bederived from non-animal sources including, but not limited to,watermelon, jack fruit, squash, coconut, green hair algae, corn and/orcotton. In some embodiments, the fibers are derived fromself-polymerization of proteins, e.g., oleosins and prolamins. In someembodiments the fibers are derived from electrospun or extrudedproteins. The fibers may form a three dimensional mesh or strandswherein each fiber may be less than 1 mm in diameter.

The adipose replica can be an emulsion comprising a solution of one ormore proteins and one or more fats suspended therein as droplets. Slowlyadding the oil phase to the aqueous phase can provide a more robustemulsion and prevents occasional failures to emulsify. Adding lecithincan, in some circumstances, destabilize a protein-stabilized emulsion,allowing for increased fat leakage when the replica is cooked. In someembodiments, the emulsion is stabilized by one or more cross-linkingenzymes into a gel. In some embodiments, the emulsion is stabilized by amatrix formed by proteins induced to gel by a heat-cool technique or acold-set-gel technique. Heating a protein-stabilized emulsion can heatdenature the proteins leading to an increase in the firmness of theadipose replica. Heating to a sufficient temperature also can reduce theviability of natural microflora by at least 100×. In some embodiments,the emulsion is stabilized by gelled protein matrix formed by acombination of one or more protein cross-linking enzymes and a heat/cooltechnique or a cold-set gel technique. After the emulsion hassufficiently cooled, but before gelling is complete, one or moreoptional ingredients can be added, such as a heme-containing protein(e.g., up to about 0.4% such as 0.15, 0.2, 0.25, 0.3, or 0.4%) to givethe adipose a more natural looking pink color and/or one of more flavorcompounds such as amino acids, sugars, thiamine, or phospholipids toprovide an improved flavor to the final product.

The one or more proteins in solution can comprise isolated and purifiedproteins, e.g. a purified pea albumin enriched fraction, a purified peaglobulin enriched fraction, a purified Moong bean 8S globulin enrichedfraction, and/or a Rubisco enriched fraction. In other embodiments, theone or more fats are derived from plant-derived oils (a rice bran oil orcanola oil). See, e.g., Section III C. In some cases the compositioncomprises a cross-linking enzyme such as a transglutaminase, lysyloxidase, or other amine oxidase. Thus, in some embodiments, an adiposetissue replica can be made by isolating and purifying one or moreproteins; preparing a solution comprising one or more proteins;emulsifying one or more fats in the solution; and stabilizing thesolution into a gelled emulsification with one or more cross-linkingreagents.

In some embodiments, the fat replica is a high fat emulsion comprising aprotein solution of purified pea albumin emulsified with 40-80% ricebran oil, stabilized with 0.5-5% (wt/vol) transglutaminase into a gel.

In some embodiments, the fat replica is a high fat emulsion comprising aprotein solution of isolated moong bean 8S globulin emulsified with40-80% rice bran oil or 40-80% canola oil, stabilized with 0.5-5%(wt/vol) transglutaminase into a gel.

The fat can be isolated from plant tissues and emulsified. Theemulsification can utilize high-speed blending, homogenization, highpressure homogenization, sonication, shearing, agitation or temperaturechanges. The lipid suspension can be sonicated or extruded to furtheralter the properties of the lipid in the solution. At this point, insome embodiments other components of the consumable are added to thesolution followed by a gelling agent. In some embodiments crosslinkingagents (e.g. transglutaminase or lysyl oxidase) are added to bind thecomponents of the consumable. In other embodiments the gelling agent isadded and the lipid/gel suspension is later combined with additionalcomponents of the consumable.

Control of Melting Point by Control of Fat Composition

The process of cooking meat is integral to the experience of using andenjoying meat. One important property of meat is that as the meat isheated, fats are released from the meat, which lubricates the cookingsurface and increases heat transfer and is a component of the visual,aural and olfactory experience of cooking meat. The amount of fat thatis released rather than retained during cooking varies with cookingtemperature and contributes to the visual, aural and olfactoryexperience of cooking meat.

The composition and ratio of fatty acids in triglycerides andphospholipids, along with the ratio of phospholipid headgroups,contribute to the generation of distinct flavor profiles of cooked meat.For example, increased levels of phosphatidylcholine andphosphatidylethanolamine in fat provide a more intense beef flavor. Asdiscussed above, the flavor of meat replicas can be modified by varyingthe ratios and type of different oils, and phospholipids that comprisethe meat replica. For example, the flavor of the cooked meat replica canbe controlled by varying the amount of phospholipids, sterols and lipids(e.g., 0.2-1% wt/wt). In one embodiment, the flavor of the cooked meatreplica can be controlled by varying the ratio of different phospholipidheadgroups.

In some embodiments, the phospholipids comprise a plurality ofamphipathic molecules comprising fatty acids, glycerol and polar groups.See, e.g., Section III C for examples of fatty acids, phospholipids,polar groups, and sterols associated with phospholipids. See alsosection III C for examples of useful plant oils.

In different cuts of meat, the fat has different properties, rangingfrom the structurally important nature of fat in bacon to the softmelting behavior of the marbling fat in Wagyu beef.

By controlling the melting point of adipose tissue replicas in theconsumables, it is possible to replicate the cooking experience ofdifferent meat types. For example, adipose tissue replicas created fromfats with a melting point of 23° C. to 27° C. can have melting pointsanalogous to adipose tissue from wagyu beef; adipose tissue replicascreated from fats with a melting point of 35° C. to 40° C. can havemelting points analogous to adipose tissue from regular ground beef; andadipose tissue replicas created from fats with a melting point of 36° C.to 45° C. can have melting points analogous to adipose tissue frombacon. Adipose tissue replicas can be created and incorporated intoconsumables such that a ratio of fat which is released and the ratio offat which is retained by adipose tissue replica during cooking issimilar to the fat properties of meat, e.g. from ground beef.

In some embodiments, the fat release temperature of a fat replicas canbe controlled by mixing different ratios of vegetable oils containingtriacylglycerides and phospholipids (e.g., lecithin). The melting pointof fats is governed by the chemical composition of fatty acids. Ingeneral, fats comprising saturated fatty acids (e.g., C10:0, C12:0,C14:0, C16:0, C18:0, C20:0, C22:0) are solid at refrigerationtemperatures (e.g., about 1° C. to about 5° C. and at room temperature(e.g., about 20° C. to 25° C.). By controlling the fat releasetemperature upon cooking in a adipose tissue replica, the firmness of aadipose tissue replica during refrigeration (e.g., about 1.5° C. toabout 4° C.) and at ambient temperature (e.g., about 20° C. to 25° C.)can be controlled. Fats comprising monounsaturated fatty acids (e.g.,C16:1 or C18:1) are generally solid at refrigeration temperatures andliquid at room temperatures. Fats comprising polyunsaturated fatty acids(e.g., C18:2, C18:3, C20:5, or C22:6) are generally liquid atrefrigeration temperatures and at room temperatures. For example, virgincoconut oil melts at about 24° C. while hydrogenated coconut oil meltsat 36-40° C.

For example, adipose tissue replicas containing triglycerides andphospholipids that are liquid at room temperature (about 20° C. to 25°C.) will be softer than adipose tissue replicas containing triglyceridesand phospholipids that are solid at refrigeration temperatures.

Adipose tissue replicas can contain oils from a single or multiplesources that are liquid at both refrigeration and ambient roomtemperatures (e.g., canola oil, sunflower oil, and/or hazelnut oil). Inone embodiment, a adipose tissue replica contains oils from single ormultiple sources that are solid at refrigeration temperature, but liquidat room temperature (e.g., olive oil, palm oil, and/or rice brain oil).In one embodiment, a adipose tissue replica contains oils from single ormultiple sources that are solid at room temperature but liquid atmouth-temperatures (about 37° C.) (e.g., palm kernel oil, coconut oil,and/or cocoa butter. In one embodiment, a adipose tissue replicacontains oils from single or multiple sources that are solid at mouthtemperature (about 37° C.) (e.g., oil from mango butter).

In one embodiment, a adipose tissue replica includes triglycerides andphospholipids with a high ratio of saturated fatty acids, and is firmerthan a adipose tissue replica containing a higher ratio ofmonounsaturated and polyunsaturated triglycerides and lipids. Forexample, a adipose tissue replica containing sunflower oil is softerthan a adipose tissue replica containing cocoa butter. Adipose tissuereplicas can be formed with 0%, 0.18%, 1.6%, or 2.4% wt/v Rubisco with70%, 80%, or 90% v/v sunflower or cocoa butter. Each adipose tissuereplica that contained cocoa butter was firmer than the replicas thatwere formed with sunflower oil.

In one embodiment, a adipose tissue replica made as a stable emulsion ofmoong bean 8S protein with sunflower oil is softer than a adipose tissuereplica made as a stable emulsion of moong bean 8S protein and cocoabutter. Adipose tissue replicas formed with 2%, 1%, or 0.5% wt/v moongbean 8S protein with 70%, 80%, or 90% v/v sunflower or cocoa butters.Each adipose tissue replica that contained cocoa butter was firmer thanthe replicas that were formed with sunflower oil.

In one embodiment, a adipose tissue replica made as a stable emulsion ofmoong bean 8S protein with canola oil is softer than a adipose tissuereplica made as a stable emulsion of moong bean 8S protein with an equalmixture of coconut, cocoa, olive, and palm oils. Adipose tissue replicascan be formed with 1.4% wt/v moong bean 8S protein with 50%, 70%, or 90%v/v sunflower or a mixture of oils. Each adipose tissue replica thatcontained a mixture of oils was firmer than the replicas that wereformed with sunflower oil.

In one embodiment, an adipose tissue replica made as a stable emulsionof soy proteins with sunflower oil is softer than a adipose tissuereplica made as a stable emulsion of soy proteins and cocoa butter.Adipose tissue replicas were formed with 0.6%, 1.6%, or 2.6% wt/v Soywith 50%, 70%, 80%, or 90% v/v sunflower or mixture of oils. Eachadipose tissue replica that contained a mixture of oils was firmer thanthe replicas that were formed with sunflower oil.

In some embodiments, the adipose tissue replicas comprising 0%, 0.18%,1.6%, and 2.4% wt/v Rubisco with 70%, 80%, and 90% v/v cocoa butter aresolid at room temperature but melt at about mouth temperature. In someembodiments, the adipose tissue replicas comprising 0.6%, 1.6%, and 2.6%wt/v soy with 50%, 70%, 80%, and 90% v/v cocoa butter are solid at roomtemperature but melt at about mouth temperature. In some embodiments,the adipose tissue replicas comprising 1.4% wt/v moong bean 8S proteinwith 50%, 70%, and 90% v/v of an equal mixture of coconut, cocoa, olive,and palm oil is solid at room temperature but melts at about mouthtemperature. In one embodiment, the melting temperature of adiposetissue replicas will be similar to beef fat. In some embodiments the fatreplicas comprise oils with a 1:1 ratio of saturated to unsaturatedfatty acids. In some embodiments, the adipose tissue replica containsequal amounts of cocoa and mango butters. In some embodiments, theadipose tissue replica contains equal amounts of coconut oil, cocoabutter, olive oil and palm oil.

In one embodiment, a adipose tissue replica that comprises triglyceridesand phospholipids will contain a ratio of fatty acids similar to thatfound in beef (C14:0 0-5% wt/wt, C16:0 0-25%, C18:0 0-20%, C18:1 0-60%,C18:2 0-25%, C18:3, 0-5%, C20:4 0-2%, and C20:6 0-2%). For example theadipose tissue replica may comprise equal proportions of olive oil,cocoa butter, coconut oil and mango butter. In another example, theadipose tissue replica may comprise equal proportions of olive oil andrice brain oil.

In one embodiment, the melting temperature of adipose tissue replicaswill be similar to Wagyu beef fat. In some embodiments the fat replicascomprise oils with a 1:2 ratio of saturated to unsaturated fatty acids(e.g., for example 1 part coconut oil to 2 parts sunflower oil). In someembodiments, the adipose tissue replica contains equal amounts of oliveoil, rice bran oil, cocoa butter and mango butter.

I. Connective Tissue Replica

Animal connective tissue provides key textural features that are animportant component of the experience of eating meat. Accordingly, thepresent invention provides a composition derived from non-animal sourceswhich recapitulates key features of animal connective tissue. Thepresent invention additionally provides a meat substitute product thatcomprises a composition derived from non-animal sources, whichrecapitulates important textural and visual features of animalconnective tissue. Such compositions will be labeled herein as“connective tissue replicas”. In some embodiments, the connective tissuereplica and/or meat substitute product comprising the connective tissuereplica are partially derived from animal sources.

Animal connective tissue can generally be divided into fascia-type andcartilage-type tissue. Fascia-type tissue is highly fibrous, resistantagainst extension (has high elastic modulus), and has a high proteincontent, a moderate water content (ca. 50%), and low-to-no fat andpolysaccharide content. Accordingly, the present invention provides aconnective tissue replica that recapitulates key features of fascia typetissue. In some embodiments, the connective tissue replica comprisesabout 50% protein by total weight, about 50% by liquid weight, and has alow fat and polysaccharide component.

The fibrous nature of fascia type connective tissue is largely comprisedof collagen fibers. Collagen fibers are observed to be cord or tapeshaped species, 1-20 microns wide. These fibers consist of closelypacked thin collagen fibrils 30 to 100 nanometers thick. These fibrilsalso associate into elastic and reticular fibrous networks withindividual fibers that may be 200 nanometers thick.

In one embodiment, the fascia-type connective tissue replica consists ofa fibrous or fibrous-like structure which can consist of proteins. Insome embodiments, the protein content is derived from non-animal source(e.g., a plant source, algae, bacteria, or fungi, see e.g., SectionsIIIA and B). In some embodiments, the isolated proteins account for 50%,60%, 70%, 80%, or 90% or more of the protein content by weight. In someembodiments, multiple isolated proteins are isolated and purifiedseparately and account for the total protein content.

In fascia-type connective tissue, the prolamin family of proteins,individually or combinations thereof, demonstrates suitability for theprotein component because they are highly abundant, similar in globalamino acid composition to collagen (high fraction of proline andalanine), and amenable to processing into films. In some embodiments,the prolamin family proteins are selected from the group consisting ofzein (found in corn), hordein from barley, gliadin from wheat, secalin,extensins from rye, kafirin from sorghum, or avenin from oats. In someembodiments, the one or more isolated and purified proteins is zein. Insome embodiments, other proteins can be used to supplement prolamins inorder to achieve targets specifications for physicochemical andnutritional properties. See, the list in Sections III A and B, includingany major seed storage proteins, animal-derived or recombinant collagen,or extensins (hydroxyproline-rich glycoproteins abundant in cell wallse.g. Arabidopsis thaliana, monomers of which are “collagen-like”rod-like flexible molecules).

The proteins can be freeze-dried and milled and combined with one ormore other ingredients (e.g., wheat gluten, fiber such as bamboo fiber,or soy protein isolate).

The fibrous or fibrous-like structures can be formed by extrusion. Insome embodiments extrusion are conducted using Leistritz Nano-16twin-screw co-rotating extruder (American Leistritz Extruder Corp. USA,Sommerville, N.J.). Active heating and cooling of the barrel section isused to optimize the mechanical properties, extent of puffing and watercontent of fibers. For example, water content can be adjusted to about50% to make a hard connective tissue replica. Protein feed and liquidare added separately: protein is feed by a volumetric plunger feeder,and liquid is added into the barrel through a high pressure liquidinjection system. In some examples, extrusion parameters were: screwspeed 200 rpm, product temperature at the die of 120° C., feed rate of2.3 g/min, and water-flow rate of 0.7 g/min. Product temperature at thedie during extrusion is measured by a thermocouple.

The fibrous or fibrous-like structures can be formed by extrusionthrough filament and multi-filament dies to produce fibrous structures.In some embodiments, dies incorporating multiple different orifice sizesin the range 10-300 microns can be used to create mixed fibrous tissuereplicas with precise control over dimensions and compositions offibers. Fibers of different sizes can be incorporated into thecompositions to control the properties of the compositions.

Electrospinning can be used to create fibers in the <1-10 micron range.In some embodiments, electrospinning is used to create fibers in the<1-10 micron diameter range. For example, a concentrated solution ofmoong bean globulin (140 mg/ml) containing 400 mM sodium chloride can bemixed with solution of poly(vinyl alcohol) or poly(ethylene oxide) (9%w/v) to obtain mixed solutions with 22.5 mg/ml moong bean globulin and6.75% w/v of the respective polymer. The resulting solution is slowly(for example, at 3 μl/min) pumped, using a syringe pump, from a 5 mlsyringe through a Teflon tube and a blunted 21 gauge needle. The needleis connected to a positive terminal of a high voltage supply (forexample, Spellman CZE 30 kV) and fixed 20-30 cm from a collectionelectrode. Collection electrode is an aluminum drum (ca. 12 cm long, 5cm in diameter) that is wrapped in aluminum foil. The drum is attachedto a spindle that is rotated by an IKA RW20 motor at about 600 rpm. Thespindle is connected to a ground terminal of the high voltage supply.Protein/polymer fiber accumulate on foil and, after electrospinning iscompleted, are removed from foil and added to tissue replicas.

The dimension and composition of the fibers produced by the methods ofthe invention has an effect upon the taste, texture, and mechanicalproperties of the tissue replicas. Tissues comprising between 1 and 50%of the fibers in the <1-10 micron range, and between 10 and 50% offibers in the 10-300 micron range most closely approach animalconnective tissues in terms of taste, mouthfeel and mechanicalproperties.

Cartilage-type tissue is macroscopically homogenous, resistant againstcompression, has higher water content (up to 80%), lower protein(collagen) content, and higher polysaccharide (proteoglycans) contents(ca. 10% each). Compositionally, cartilage-type connective tissuereplicas are similar to fascia-type tissue replicas with the relativeratios of each adjusted to more closely mimic ‘meat’ connective tissue.During extrusion, water content can be adjusted to about 60% to make asoft connective tissue replica.

Methods for forming cartilage-type connective tissue are similar tothose for fascia-type connective tissue, but methods that produceisotropic non-fibrous gels are preferred.

A connective tissue replica can be made by isolating and purifying oneor more proteins; and precipitating the one or more proteins, whereinthe precipitating results in the one or more proteins forming physicalstructures approximating the physical organization of connective tissue.The precipitating can comprise solubilizing the one or more proteins ina first solution; and extruding the first solution into a secondsolution, wherein the one or more proteins is insoluble in the secondsolution, wherein the extruding induces precipitation of the one or moreproteins.

In some embodiments some or all of the components of the consumable aresuspended in a gel (e.g., a proteinacious gel). In various embodimentsthe gel can be a hydrogel, an organogel, or a xerogel. The gel can bethickened using an agent based on polysaccharides or proteins. Forexample fecula, arrowroot, cornstarch, katakuri starch, potato starch,sago, tapioca, alginin, guar gum, locust bean gum, xanthan gum,collagen, egg whites, furcellaran, gelatin, agar, carrageenan,cellulose, methylcellulose, hydroxymethylcellulose, acadia gum, konjac,starch, pectin, amylopectin or proteins derived from legumes, grains,nuts, other seeds, leaves, algae, bacteria, of fungi can be used aloneor in combination to thicken the gel, forming an architecture orstructure for the consumable. Enzymes that catalyze reactions leading tocovalent crosslinks between proteins can also be used alone or incombination to form an architecture or structure for the consumable. Forexample transglutaminase, tyrosinases, lysyl oxidases, or other amineoxidases (e.g. Pichia pastoris lysyl oxidase (PPLO)) can be used aloneor in combination to form an architecture or structure for theconsumable by crosslinking the component proteins. In some embodiments,multiple gels with different components are combined to form theconsumable. For example a gel containing a plant-derived protein can beassociated with a gel containing a plant-derived fat. In someembodiments fibers or strings of proteins are oriented parallel to oneanother and then held in place by the application of a gel containingplant based fats.

The compositions of the invention can be puffed or expanded by heating,such as frying, baking, microwave heating, heating in a forced airsystem, heating in an air tunnel, and the like, according to methodswell known in the art.

In some embodiments multiple gels with different components are combinedto form the consumable. For example a gel containing a plant-derivedprotein can be associated with a gel containing a plant-derived fat. Insome embodiments fibers or strings of proteins are oriented parallel toone another and then held in place by the application of a gelcontaining plant based fats.

J. Omissions from the Compositions

Because the consumable can be put together from defined ingredients,which may themselves be isolated and purified, it is possible to produceconsumables that do not contain certain components. This, in some cases,allows for the production of consumables that are lacking ingredientsthat may be not desirable to consumers (e.g., proteins that some humansare allergic to can be omitted or additives). In some embodiments, theconsumable contains no animal products. In some embodiments theconsumable contains no or less than 1% wheat gluten. In some embodimentsthe consumable contains no methylcellulose. In some embodiments theconsumable contains no carrageenan. In some embodiments the consumablecontains no caramel color. In some embodiments the consumable containsno Konjac flour. In some embodiments the consumable contains no gumarabic (also known as acacia gum). In some embodiments the consumablecontains no wheat gluten. In some embodiments the consumable contains nosoy protein isolate. In some embodiments the consumable contains notofu. In some embodiments the consumable contains less than 5%carbohydrates. In some embodiments the consumable contains less than 1%cellulose. In some embodiments the consumable contains less than 5%cellulose. In some embodiments the consumable contains less than 5%insoluble carbohydrates. In some embodiments the consumable containsless than 1% insoluble carbohydrates. In some embodiments the consumablecontains no artificial color. In some embodiments the consumablecontains no artificial flavorings.

In some embodiments the consumable contains one or more of the followingcharacteristics: no animal products; no methylcellulose; no carrageenan;no Konjac flour; no gum arabic; less than 1% wheat gluten; no wheatgluten; no tofu; about 5% carbohydrates; less than 5% cellulose; lessthan 5% insoluble carbohydrates; less than 1% insoluble carbohydrates;no edible colorants such as caramel color, paprika, cinnamon, beetcolor, carrot oil, tomato lycopene extract, raspberry powder, carmine,cochineal extract, annatto, turmeric, saffron, F.D&C Red No. 3, Yellownumber 5, Yellow No 6, Green No. 3, Blue No. 2, Blue No. 1, Violet No.1,FD&C Red No. 40—Allura Red AC, and/or E129 (red shade); and/or noartificial flavorings. In some embodiments the consumable contains nosoy protein isolate. In other embodiments, the consumable contains nosoy protein or protein concentrate.

In some embodiments, the muscle tissue replica additionally containsless than 10%, less than 5%, less than 1%, or less than 0.1% wheatgluten. In some embodiments, the muscle tissue replica contains no wheatgluten.

IV. Combinations of the Components

A. Meat Replicas

A meat substitute product (alternatively a meat replica) can comprisecompositions described herein. For example a meat replica can comprise amuscle replica; a fat tissue replica; and a connective tissue replica(or a sub-combination thereof). The muscle replica, adipose tissuereplica, and/or connective tissue replica can be assembled in a mannerthat approximates the physical organization of meat. In someembodiments, a binding agent such as a coacervate is used to helpbinding the replicas to each other.

The percentage of different components may also be controlled. Forexample non-animal-based substitutes for muscle, adipose tissue,connective tissue, and blood components can be combined in differentratios and physical organizations to best approximate the look and feelof meat. The various components can be arranged to insure consistencybetween bites of the consumable. The components can be arranged toinsure that no waste is generated from the consumable. For example,while a traditional cut of meat may have portions that are not typicallyeaten, a meat replicate can improve upon meat by not including theseinedible portions (e.g, bone, cartilage, connective tissue, or othermaterials commonly referred to as gristle). Such an improvement allowsfor all of the product made or shipped to be consumed, which cuts downon waste and shipping costs. Alternatively, a meat replica may includeinedible portions to mimic the experience of meat consumption. Suchportions can include bone, cartilage, connective tissue, or othermaterials commonly referred to as gristle, or materials includedsimulating these components. In some embodiments the consumable maycontain simulated inedible portions of meat products which are designedto serve secondary functions. For example a simulated bone can bedesigned to disperse heat during cooking, making the cooking of theconsumable faster or more uniform than meat. In other embodiments asimulated bone may also serve to keep the consumable at a constanttemperature during shipping. In other embodiments, the simulatedinedible portions may be biodegradable (e.g., a biodegradable plastic).

In some embodiments, a meat substitute composition comprises between10-30% protein, between 5-80% water, and between 5-70% fat, wherein thecomposition includes one or more isolated and purified proteins. Such ameat substitute can include no animal protein. In some embodiments, themeat substitute compositions comprise a transglutaminase.

In some embodiments, a meat substitute product includes a musclereplica, an adipose tissue replica, and connective tissue replica, wherethe muscle replica accounts for 40-90% of the product by weight, theadipose tissue replica accounts for 1-60% of the product by weight, andthe connective tissue replica accounts for 1-30% of the product byweight.

In some embodiments, the meat substitute product comprises 60-90% water;5-30% protein content; and 1-20% of a fat; wherein the protein contentcomprises one or more isolated and purified plant proteins.

In some embodiments the consumable contains components to replicate thecomponents of meat. The main component of meat is typically skeletalmuscle. Skeletal muscle typically consists of roughly 75 percent water,19 percent protein, 2.5 percent intramuscular fat, 1.2 percentcarbohydrates and 2.3 percent other soluble non-protein substances.These include organic acids, sulfur compounds, nitrogenous compounds,such as amino acids and nucleotides, and inorganic substances such asminerals. Accordingly, some embodiments of the present invention providefor replicating approximations of this composition for the consumable.For example, in some embodiments, the consumable is a plant-based meatreplica comprising roughly 75% water, 19% protein, 2.5% fat, 1.2%carbohydrates; and 2.3 percent other soluble non-protein substances. Insome embodiments the consumable is a plant-based meat replica comprisingbetween 60-90% water, 10-30% protein, 1-20% fat, 0.1-5% carbohydrates;and 1-10 percent other soluble non-protein substances. In someembodiments the consumable is a plant-based meat replica comprisingbetween 60-90% water, 5-10% protein, 1-20% fat, 0.1-5% carbohydrates;and 1-10 percent other soluble non-protein substances. In someembodiments the consumable is a plant-based meat replica comprisingbetween 0-50% water, 5-30% protein, 20-80%% fat, 0.1-5% carbohydrates;and 1-10 percent other soluble non-protein substances.

In some embodiments, a meat replica contains between 0.01% and 5% byweight of a heme containing protein. In some embodiments, the replicacontains between 0.01% and 5% by weight of leghemoglobin. Some meat alsocontains myoglobin, a heme containing protein, which accounts for mostof the red color and iron content of some meat. It is understood thatthese percentages can vary in meat and the meat replicas can be producedto approximate the natural variation in meat. In embodiments thatinclude a heme-containing protein and optional flavors, k-carrageenancan be used absorb some of the liquid contributed from the flavor andheme solution so the ground tissue is not excessively wet. Duringaddition of flavor heme mix solution and the k-carrageenan powder aredistributed evenly over the tissue mixture to ensure homogeneity in thefinal ground product.

It will be appreciated that when proteins are supplied as a solution,water removal techniques such as freeze-drying or spray drying canoptionally be used to concentrate the protein. The proteins may then bereconstituted in an amount of liquid that prevents the ground tissuefrom being too moist.

Additionally, in some instances, the present invention provides forimproved meat replicas, which comprise these components in unnaturalpercentages. The concentration of heme containing protein is animportant determinant of meat flavor and aroma. Thus for example a meatreplica could have a higher heme protein content than typical beef. Forexample a meat replica can be produced with a higher than typicalaverage fat content. The percentages of these components may also bealtered to increase other desirable properties.

In some instances a meat replica is designed so that, when cooked, thepercentages of components are similar to cooked meat. So, in someembodiments, the uncooked consumable has different percentages ofcomponents than uncooked meat, but when cooked, the consumable issimilar to cooked meat. For example, a meat replica may be made with ahigher than typical water content for raw meat, but when cooked in amicrowave, the resulting product has non-starch polysaccharides such asarabinoxylans, cellulose, and many other plant components such asresistant starch, resistant dextrins, inulin, lignin, waxes, chitins,pectins, beta-glucans, and oligosaccharide percentages of componentssimilar to meat cooked over a fire.

In some embodiments, the consumable is a meat replica with a lower thattypical water content for meat. In some embodiments the inventionsprovides for methods for hydrating a meat replica to cause the meatreplica to have a water content similar to meat. For example a meatreplica with a water content that would be low for meat, for example 1%,10%, 20%, 30%, 40% or 50% water, can be hydrated to roughly 75% water.Once hydrated, in some embodiments, the meat replica is then cooked forhuman consumption.

The consumable can have a protein component. In some embodiments theprotein content of the consumable is 10%, 20%, 30%, or 40%. In someembodiments the protein content of the consumable is similar to meat. Insome embodiments the protein content in the consumable is greater thanthat of meat. In some embodiments the consumable has less protein thanmeat.

The protein in the consumable can come from a variety or combination ofsources. Non-animal sources can provide some or all of the protein inthe consumable. Non-animal sources can include vegetables, non foodbiomass such as carrot tops and Miscanthus, seaweed, fruits, nuts,grains, algae, bacteria, or fungi. See, e.g, Sections III A and B. Theprotein can be isolated or concentrated from one or more of thesesources. In some embodiments the consumable is a meat replica comprisingprotein only obtained from non-animal sources.

In some embodiments protein is formed into asymmetric fibers forincorporation into the consumable. In some embodiments these fibersreplicate muscle fibers. In some embodiments the protein are spunfibers. Accordingly, the present invention provides for methods forproducing asymmetric or spun protein fibers. In some embodiments theconsumable contains a protein or proteins that have all of the aminoacids found in proteins that are essential for human nutrition. In someembodiments the proteins added to the consumable are supplemented withamino acids.

The physical organization can be a determinant of the response of themeat substitute to cooking. For example, the flavor of meat is modifiedby the size of the particles. Ground meat that has been reduced to apaste provides different flavors than more crudely ground beef uponcooking. The ability to control the relative size and orientation ofindividual tissue replicas enables the flavor and aroma profile ofconsumables to be modified during cooking. For example, muscle tissuereplicas and adipose tissue replicas provide different flavor profileswhen cooked independently or when mixed. Further changes in the flavorprofile are observed based on the method by which the different tissuereplicas are mingled.

The physical organization of the meat substitute product can bemanipulated by controlling the localization, organization, assembly, ororientation of the muscle, fat, and/or connective tissue replicasdescribed herein. In some embodiments the product is designed in such away that the replicas described herein are associated with one anotheras in meat. In some embodiments the consumable is designed so that aftercooking the replicas described herein are associated with one another asin cooked meat.

Characteristic flavor and fragrance components of meat are mostlyproduced during the cooking process by chemical reactions, thesubstrates for which are amino acids, fats and sugars which are found inplants as well as meat. Therefore in some embodiments the consumable istested for similarity to meat during or after cooking. In someembodiments human ratings, human evaluation, olfactometer readings, orGCMS measurements, or combinations thereof, are used to create anolfactory map of cooked meat. Similarly, an olfactory map of theconsumable, for instance a meat replica, can be created. These maps canbe compared to assess how similar the cooked consumable it so meat. Insome embodiments the olfactory map of the consumable during or aftercooking is similar to or indistinguishable from that of cooked orcooking meat. In some embodiments the differences are sufficiently smallas to be below the detection threshold of human perception.

In some embodiments the individual tissue replicas are assembled inlayers, sheets, blocks and strings in defined positions andorientations.

In some embodiments, the replicas are combined in the process of passingthrough the plates of a meat grinder with the holes set at less than ½inch (e.g., at ¼ inch). The grinder provides multiple functions ofreducing the particle size, providing additional mixing or working, andforming the material into cylindrical portions like what is typicallydone for ground beef. During the assembly, grinding, and forming, it isimportant to keep the replica tissues cold (e.g., 4-15° C.) to controlmicrobial growth, limit flavor reactions, and also to maintain theadipose in a solid state so discrete pieces of adipose will bemaintained through the grinding process.

Prior to grinding, the replica tissues are usually broken down in somemanner to a defined particle size. For example, in some embodiments, theindividual tissue replicas can be formed into small pieces less than 1cm in diameter or less than 5 mm in diameter before combination with theother tissue replicas. Adipose tissue can be crumbled into about 3-7 mmparticles. This is important to both the appearance of the finalmaterial and the behavior of fat leakage during cooking. This size rangeallows for a natural appearance of adipose flecks in the final rawproduct. If the adipose tissues are too small (e.g., less than 2 mm),there will be an insufficient amount of fat leaked from the product whencooked.

Soft connective tissue replicas can be broken down into pieces of about1-3 mm in length with ragged edges. If the pieces are too large (e.g.,greater than about 4 mm), the texture of the final product can be toobeady.

Sticky tissue or noodles tissue replicas, composed of amorphous or longnoodle like tissue replica pieces, respectively, and raw tissue replicascan be manually broken down into pieces about 1-3 cm in diameter.Achieving particles in this size range allows for adequate mixing andsuitable homogeneity in the final ground material.

In some embodiments, hard connective tissue replicas can be chopped tothree levels, (e.g., coarse, intermediate, and fine). Chopping to threelevels provides a greater amount of heterogeneity than a single stepchopping process, and makes the mouthfeel of the final product moresimilar to ground beef.

In formulations containing gluten an additional function of the foodgrinder is to work the gluten and develop a gluten network of alignedgluten molecules. For gluten containing formulations it is important tominimize interaction of the adipose with the gluten network. This isdone by prechilling the adipose replicas and ground tissue replicasprior to combining and also by minimizing the amount of manipulationafter the adipose is added. Overworking of adipose replicas into thegluten will break down or “shorten” the gluten network.

Finally for gluten containing formulations the patties are allowed torest at room temperature for 30 min or overnight at 4 C prior tocooking. This allows time for the gluten network to relax, giving anoverall better texture.

In some embodiments, the connective tissue replica is incorporated intothe protein solution before formation of the muscle tissue replica.

In some embodiments, the connective tissue replica is directlyincorporated into the emulsion before formation of the adipose tissuereplica.

In some embodiments, the adipose tissue replica is added to the muscletissue replica in strands and sheets to replicate the effect of“marbling,” or streaky bacon.

Mixed meat tissue replicas can increase the sense of flavors, suchflavors including, but not limited to, multiple aromatic compoundsassociated with fruity/grean bean/metallic, nutty/green, peanutbutter/musty, raw potato/roasted/earthy, vinegary, spicy/caramel/almond,creamy, sweet, fruity/stale beer,musty/nutty/coumarin/licorice/walnut/bread, coconut/woody/sweet,penetrating/sickening, minty, or toasty caramel aromas.

In some embodiments the mixed meat replicas increase the presence ofvolatile odorants, such as 2-pentyl-furan; 4-methylthiazole; ethylpyrazine; 2,3-dimethylpyrazine, acetic acid;5-methyl-2-furancarboxaldehyde; butyrolactone; 2,5-dimethyl-3-(3-methylbutyl) pyrazine; 2-cyclopentene-1-one, 2-hydroxy-3-methyl;3-acetyl-1h-pyrolline; pantolactone; 1-methyl1(H)-pyrrole-2-2carboxaldehyde; caprolactam;2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one. in someembodiments, the undesired flavors including but not limited togasoline-like, petroleum, sour/putrid/fish-like, bland/woody/yogurt,fatty/honey/citrus, pungent/sweet/caramelic and nutty/burnt greenaromas, form only in individual tissue replicas, but do not accumulatein mixed meat replicas. In some embodiments, the individual tissuereplicas increase the presence of volatile odorants including but notlimited to nonane, 2,6-dimethyl, 3-methyl 3-hexene; pyridine; acetoin;octanal; 1-hydroxy-2-propanone; and/or ethenyl pyrazine. In someembodiments the levels to which all of the above compounds accumulateduring cooking depend on the sizes of tissue replica units and how theyare mixed (coarse, fine, or blended).

In some embodiments, the mixed meat tissue replicas increase the senseof flavors including but not limited to, multiple aromatic compoundsassociated with fruity/grean bean/metallic, nutty/green, peanutbutter/musty, raw potato/roasted/earthy, vinegary, spicy/caramel/almond,creamy, sweet, fruity/stale beer,musty/nutty/coumarin/licorice/walnut/bread, coconut/woody/sweet,penetrating/sickening, minty, or toasty caramel aromas. In someembodiments, the mixed meat replicas increase the presence of volatileodorants including but not limited to phenylacetaldehyde, 1-octen-3-one,2-n-heptylfuran, 2-thiophenecarboxaldehyde, 3-thiophenecarboxaldehyde,butyrolactone, 2-undecenal, methyl-pyrazine, furfural, 2-decanone,pyrrole, 1-octen-3-ol, 2-acetylthiazole, (E)-2-octenal, decanal,benzaldehyde, (E)-2-nonenal, pyrazine, 1-hexanol, 1-heptanol, dimethyltrisulfide, 2-nonanone, 2-pentanone, 2-heptanone, 2,3-butanedione,heptanal, nonanal, 2-octanone, 1-octanol, 3-ethylcyclopentanone,3-octen-2-one, (E,E)-2,4-heptadienal, (Z)-2-heptenal, 2-heptanone,6-methyl-, (Z)-4-heptenal, (E,Z)-2,6-nonadienal, 3-methyl-2-butenal,2-pentyl-furan, thiazole, (E, E)-2,4-decadienal, hexanoic acid,1-ethyl-5-methylcyclopentene, (E,E)-2,4-nonadienal, (Z)-2-decenal,dihydro-5-pentyl-2(3H)-furanone, trans-3-nonen-2-one,(E,E)-3,5-octadien-2-one, (Z)-2-octen-1-ol,5-ethyldihydro-2(3H)-furanone, 2-butenal, 1-penten-3-ol, (E)-2-hexenal,formic acid, heptyl ester, 2-pentyl-thiophene, (Z)-2-nonenal,2-hexyl-thiophene, (E)-2-decenal, 2-ethyl-5-methyl-pyrazine,3-ethyl-2,5-dimethylpyrazine, 2-ethyl-1-hexanol, thiophene,2-methyl-furan, pyridine, butanal, 2-ethyl-furan, 3-methyl-butanal,trichloromethane, 2-methyl-butanal, methacrolein, 2-methyl-propanal,propanal, acetaldehyde, 2-propyl-furan, dihydro-5-propyl-2(3H)-furanone,1,3-hexadiene, 4-decyne, pentanal, 1-propanol, heptanoic acid,trimethyl-ethanethiol, 1-butanol, 1-penten-3-one, dimethyl sulfide,2-ethyl furan, 2-pentyl-thiophene, 2-propenal, 2-tridecen-1-ol,4-octene, 2-methyl thiazole, methyl-pyrazine, 2-butanone,2-pentyl-furan, 2-methyl-propanal, butyrolactone, 3-methyl-butanal,methyl-thiirane, 2-hexyl-furan, butanal, 2-methyl-butanal,2-methyl-furan, furan, octanal, 2-heptenal, 1-octene, formic acid heptylester, 3-pentyl-furan, and 4-penten-2-one. In some embodiments thelevels to which all of the above compounds accumulate during cookingdepend on the sizes of tissue units and how they are mixed (coarse,fine, or blended).

The production of volatile odorants can be enhanced when the adipose,muscle and connective tissue replicas are contacting one another. Insome embodiments, the production of volatile odorants is enhanced whenthe adipose, muscle and connective tissues are intimately mixed with amean size of the individual tissue replicas of 5 mm. In someembodiments, the production of volatile odorants is enhanced when thefat, muscle and connective tissues are intimately mixed with mean sizeof the individual tissue replicas of 2 mm. In some embodiments, saidproduction of volatile odorants is enhanced when the fat, muscle andconnective tissue replicas are intimately mixed with mean size of theindividual tissue replicas of 1 mm.

In some embodiments, the meat substitute is optimized for particularcooking methods (optimized for cooking in a microwave oven, or optimizedfor cooking in a stew).

In some embodiments, the meat substitute is optimized for dehydration.

In some embodiments, said meat substitute is optimized for fastrehydration upon exposure of the dehydrated meat replica to water.

In some embodiments, said meat substitute is optimized for use asemergency, camping or astronaut food.

The methods described herein can be used to provide a meat replica withdefined cooking characteristics to allow the production of meat replicasthat are optimized for particular cooking techniques. For example, stewsrequire slow cooking to gelatinize the connective tissue in the meat,whereas meat replicas can be designed wherein the connective tissuereplica is more easily gelatinized thus allowing stews to be preparedquickly.

B. Indicators of Cooking Meat

The consumable can include compositions which can indicate that theconsumable is cooking or has cooked. The release of odorants uponcooking is an important aspect of meat consumption. In some embodiments,the consumable is a meat replica entirely composed of non-animalproducts that when cooked generates an aroma recognizable by humans astypical of cooking beef. In some embodiments, the consumable when cookedgenerates an aroma recognizable by humans as typical of cooking pork,bacon, chicken, lamb, fish, or turkey. In some embodiments theconsumable is a meat replica principally or entirely composed ofingredients derived from non-animal sources, with an odorant that isreleased upon cooking or that is produced by chemical reactions thattake place upon cooking. In some embodiments the consumable is a meatreplica principally or entirely composed of ingredients derived fromnon-animal sources, containing mixtures of proteins, peptides, aminoacids, nucleotides, sugars and polysaccharides and fats in combinationsand spatial arrangements that enable these compounds to undergo chemicalreactions during cooking to produce odorants and flavor-producingcompounds.

In some embodiments, the consumable is a meat replica principally orentirely composed of ingredients derived from non-animal sources, with avolatile or labile odorant that is released upon cooking.

In some embodiments, the indicator is a visual indicator that accuratelymimics the color transition of a meat product during the cookingprogression. The color transition can be, for example, from red tobrown, from pink to white or tan, or from a translucent to opaque colorduring the cooking progression.

In some embodiments, the indicator is an olfactory indicator thatindicates cooking progression. In one embodiment, the olfactoryindicator is one or more volatile odorants released during cooking.

In some embodiments, the indicator comprises one or more isolated,purified iron-containing proteins. In some embodiments, the one or moreisolated, purified iron-containing proteins (e.g., a heme-containingprotein, see section III B) is in a reduced state before cooking. Insome embodiments, the one or more isolated and purified iron carryingproteins in a reduced or oxidized state has a similar UV-VIS profile toa myoglobin protein derived from an animal source when in an equivalentreduced or oxidized state. The Aquifex aeolicus hemoglobin has a peakabsorbance wavelength at 413 nm; the Methylacidiphilum infernorumhemoglobin has a peak absorbance wavelength at 412 nm; the Glycine maxleghemoglobin has a peak absorbance wavelength at 415 nm; the Hordeumvulgare and Vigna radiata non-symbiotic hemoglobins each have a peakabsorbance wavelength at 412 nm. The Bos taurus myoglobin has a peakabsorbance wavelength at 415 nm.

In some embodiments, the difference between the peak absorbancewavelength of the one or more isolated and purified iron-containingproteins and the peak absorbance wavelength of myoglobin derived from ananimal source is less than 5%.

Odorants released during cooking of meat are generated by reactions thatcan involve as reactants fats, protein, amino acids, peptides,nucleotides, organic acids, sulfur compounds, sugars and othercarbohydrates. In some embodiments the odorants that combine during thecooking of meat are identified and located near one another in theconsumable, such that upon cooking of the consumable the odorantscombine. So, in some embodiments, the characteristic flavor andfragrance components are produced during the cooking process by chemicalreactions involving amino acids, fats and sugars found in plants as wellas meat. So, in some embodiments, the characteristic flavor andfragrance components are mostly produced during the cooking process bychemical reactions involving one or more amino acids, fats, peptides,nucleotides, organic acids, sulfur compounds, sugars and othercarbohydrates found in plants as well as meat.

Some reactions that generate odorants released during cooking of meatcan be catalyzed by iron, in particular the heme iron of myoglobin. Thusin some embodiments, some of the characteristic flavor and fragrancecomponents are produced during the cooking process by chemical reactionscatalyzed by iron. In some embodiments, some of the characteristicflavor and fragrance components are produced during the cooking processby chemical reactions catalyzed by heme. In some embodiments, some ofthe characteristic flavor and fragrance components are produced duringthe cooking process by chemical reactions catalyzed by the heme iron inleghemoglobin. In some embodiments, some of the characteristic flavorand fragrance components are produced during the cooking process bychemical reactions catalyzed by the heme iron in a heme protein. Forexample, hemeproteins (e.g., from Aquifex aeolicusm, Methylacidiphiluminfernorum, Glycine max, Hordeum vulgare, or Vigna radiate) provide asignificantly different profile of volatile odorants when heated in thepresence of cysteine and glucose than any subset of the three componentswhen analysed by GC-MS. Volatile flavor components that are increasedunder these conditions include but are not limited to furan, acetone,thiazole, furfural, benzaldehyde, 2-pyridinecarboxaldehyde,5-methyl-2-thiophenecarboxaldehyde, 3-methyl-2-thiophenecarboxaldehyde,3-thiophenmethanol and decanol. Under these conditions, cysteine andglucose alone or in the presence of iron salts such as ferrous glucanateproduced a sulfurous, odor but addition of heme proteins reduced thesulfurous odor and replaced it with flavors including but not limited tochicken broth, burnt mushroom, molasses, and bread.

Additionally, a hemeprotein (e.g., from Aquifex aeolicus,Methylacidiphilum infernorum, Glycine max, Hordeum vulgare, or Vignaradiata) when heated in the presence of ground chicken increasedspecific volatile odorants that are elevated in beef compared to chickenwhen analyzed by GC-MS. Volatile flavor components that are increasedunder these conditions include but are not limited to propanal, butanal,2-ethyl-furan, heptanal, octanal, trans-2-(2-pentenyl)furan,(Z)-2-heptenal (E)-2-octenal pyrrole, 2,4-dodecadienal, 1-octanal, or(Z)-2-decenal 2-undecenal.

C. Color Indicators

The color of meat is an important part the experience of cooking andeating meat. For instance, cuts of beef are of a characteristic redcolor in a raw state and gradually transition to a brown color duringcooking. As another example, white meats such as chicken or pork have acharacteristic pink color in their raw state and gradually transition toa white or brownish color during cooking. The amount of the colortransition is used to indicate the cooking progression of beef andtitrate the cooking time and temperature to produce the desired state ofdone-ness. In some aspects, the invention provides a non-meat based meatsubstitute product that provides a visual indicator of cookingprogression. In some embodiments, the visual indicator is a colorindicator that undergoes a color transition during cooking. In someembodiments, the color indicator recapitulates the color transition of acut of meat as the meat progresses from a raw to a cooked state. In moreembodiments, the color indicator colors the meat substitute product ared color before cooking to indicate a raw state and causes the meatsubstitute product to transition to a brown color during cookingprogression. In other embodiments, the color indicator colors the meatsubstitute product a pink color before cooking to indicate a raw stateand causes the meat substitute product to transition to a white or browncolor during cooking progression.

The main determinant of the nutritional definition of the color of meatis the concentration of iron carrying proteins in the meat. In theskeletal muscle component of meat products, one of the mainiron-carrying proteins is myoglobin. As described above, the myoglobincontent of varies from under 0.05% in the white meat of chicken to1.5-2.0% in old beef. So, in some embodiments, the consumable is a meatreplica which comprises an iron-carrying protein (e.g., aheme-containing protein). In some embodiments, the meat replicacomprises about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%,about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%,about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%,about 1.7%, about 1.8%, about 1.9%, about 2%, or more than about 2% ofan iron-carrying protein (e.g., a heme-containing protein) by dry weightor total weight. In some cases, the iron carrying protein has beenisolated and purified from a source. In other cases, the iron carryingprotein has not been isolated and purified. In some cases, the source ofthe iron-carrying protein is an animal source, or a non-animal sourcesuch as a plant, fungus, or genetically modified organisms such as,e.g., plant, algae, bacteria or fungus. In some cases, the iron-carryingprotein is myoglobin. In some embodiments the consumable is a plantbased meat replica that has animal myoglobin added. So, for example areplica of young beef can have about 0.4-1% myoglobin. In someembodiments the consumable is a plant based meat replica that has aleghemoglobin or a cytochrome added. So, for example, a replica of youngbeef can have about 0.4-1% leghemoglobin or cytochrome.

Another example of iron-carrying proteins is hemoglobin, theiron-containing oxygen-binding protein in the red blood cells ofvertebrates. Hemoglobin is similar in color to myoglobin. In someembodiments the invention provides methods of saving and recycling bloodfrom animal farming to supplement the color of a consumable. Forexample, blood is saved from a slaughter house, and hemoglobin from theblood is used to enhance the color of a consumable. In some aspects theconsumable is a plant-based meat replica containing hemoglobin.

Additional iron containing proteins exist in nature. In some embodimentsthe consumable comprises an iron containing protein that is notmyoglobin. In some embodiments the consumable does not containmyoglobin. In some embodiments the consumable does not containhemoglobin. In some embodiments the consumable is a meat replica thatcomprises an iron containing protein other than myoglobin or hemoglobin.See, for example, Section III B for examples of heme-containingproteins, as well as FIG. 3. For example, in some embodiments theconsumable comprises a hemoprotein (e.g., a hemoglobin, myoglobin,neuroglobin, cytoglobin, leghemoglobin, non-symbiotic hemoglobin, Hell'sgate globin I, a bacterial hemoglobin, a ciliate myoglobin, or aflavohemoglobin).

Leghemoglobin, similar in structure and physical properties tomyoglobin, is readily available as an unused by-product of commoditylegume crops (e.g., soybean or pea). The leghemoglobin in the roots ofthese crops in the US exceeds the myoglobin content of all the red meatconsumed in the US.

In some embodiments, the consumable is a meat replica principally orentirely composed of ingredients derived from non-animal sources, andcontaining a heme protein (e.g., a leghemoglobin or member of the globinprotein family). For example, a meat replica can be principally orentirely composed of ingredients derived from non-animal sources,including a muscle tissue replica, an adipose tissue replica, aconnective tissue replica, and a heme protein. In some embodiments theconsumable is a meat replica principally or entirely composed ofingredients derived from non-animal sources, with a high iron contentfrom a heme protein. In some embodiments the iron content is similar tomeat. In some embodiments the consumable has the distinctive red colorof meat, such color provided by leghemoglobin.

A heme protein (e.g., a heme-containing protein described in Section IIIB) can be used as an indicator that the consumable is finished cooking.So, one embodiment of the invention is a method for cooking a consumablecomprising detecting leghemoglobin, which has migrated from the interiorof the consumable to the surface when the product is cooked. Anotherembodiment of the invention is a method for cooking a consumablecomprising detecting the change in color of from red to brown when theproduct is cooked.

In some embodiments, the increased shelf life is provided by anextension of the lifetime of the desired red color of food products(e.g., a non-meat based meat substitute).

In one embodiment, this invention provides hemoproteins that provide adesired color to non-meat meat substitutes. In some embodiments, thehemoproteins are derived from a non-animal source such as a plant,fungus, or genetically modified organisms such as, e.g., plant, algae,bacteria or fungus. See, e.g., section III B. In some embodiments thelife time of the hemoproteins is extended by treatment with meat shelflife extenders.

In some embodiments, the meat shelf life extenders are selected from agroups consisting of carbon monoxide, nitrites, sodium metabisulfite,Bombal, rosemary extract, green tea extract, catechins and otheranti-oxidants.

In one embodiment, this invention provides hemoproteins that provide adesired flavor profiles to food products (e.g., non-meat meatsubstitutes). In some embodiments, the ability of the hemoproteins togenerate the desired flavor profile is similar to that of myoglobin.

In some embodiments, the life time of the ability of the hemoproteins togenerate the desired flavor profile is 10%, 20%, 30% 50%, or 100% ormore greater than that of myoglobin.

D. Food Products Comprising Isolated, Purified Heme Proteins

In some embodiments, heme proteins described herein are added to meat ora consumable described herein to enhance the properties of the meat orconsumable. For example, a heme protein containing solution can beinjected into raw (e.g., raw white meat) or cooked meat to improve theorganoleptic properties of the meat during cooking adding a “beefy”flavor (e.g., to white meats such as chicken).

In another example, a heme protein solution can be dripped over meat ora consumable of the invention to enhance appearance. In one embodiment,advertising, photography, or videography of food products such as meator a meat substitute can be enhanced with a heme protein.

In another embodiment, a heme protein is added to the consumable as aniron supplement.

In one application of the invention, hemeproteins may be used as fooddyes. In one embodiment, the heme proteins may be used as a safe,digestible replacement for FD&C Red No. 40—Allura Red AC, E129 (redshade) in a variety of applications. A non-limiting list of suchpotential uses would include making pictures, especially in forms suchas body-painting or as theatrical blood.

In some embodiments, the present invention provides methods forobtaining hemeproteins (e.g., leghemoglobin) from a plant. Leghemoglobincan be obtained from a variety of plants. Various legumes species andtheir varieties (e.g., soybean, fava bean, lima bean, cowpeas, Englishpeas, yellow peas, Lupine, kidney beans, garbanzo beans, peanuts,Alfalfa, Vetch hay, clover, Lespedeza, or pinto bean) containnitrogen-fixing root nodules in which leghemoglobin has a key role incontrolling oxygen concentrations (for example root nodules from a peaplant). In one embodiment leghemoglobin protein is purified from rootnodules of legume plants (e.g., soybeans, favabeans, or peas) usingion-exchange chromatography. In an embodiment, leghemoglobin is purifiedfrom soybean, favabean or sweet pea root nodules.

Plants can be grown using standard agricultural methods, with theexception that, in some instances, fertilizer is not applied and soil isenriched in natural nitrogen-fixing bacteria from the Rhizobium genus.Either whole roots or root nodules can be harvested and lysed, forexample in 20 mM potassium phosphate pH 7.4, 100 mM potassium chlorideand 5 mM EDTA using grinder-blender. During this process, leghemoglobinis released into the buffer. Root-nodule lysate containing leghemoglobincan be cleared from cell debris by filtration through 5 μm filter. Insome embodiments, filtration is followed by centrifugation (7000 g, 20min). Clarified lysate containing leghemoglobin is then filtered through200 nm filter and applied onto anion-exchange chromatography column(High Prep Q; High Prep DEAE, GE Healthtcare) on fast protein liquidchromatography machine (GE Healthcare). Leghemoglobin is collected inthe flowthrough fraction and concentrated over 3 kDa filtration membraneto a desired concentration. Purity (partial abundance) of purifiedleghemoglobin is analyzed by SDS-PAGE gel: in lysate leghemoglobin ispresent at 20-40%, while after anion-exchange purification it is presentat 70-80%. In another embodiment, soybean leghemoglobin flowthrough fromanion-exchange chromatography is applied onto size-exclusionchromatography (Sephacryl S-100 HR, GE Healthcare). Soybeanleghemoglobin is eluted as two fractions corresponding to dimer andmonomer species. Purity (partial abundance) of leghemoglobin wasanalyzed by SDS-PAGE and determined to be ˜90-100%.

Proteins in legume root-nodule lysate can be transferred into 10 mMsodium carbonate pH 9.5, 50 mM sodium chloride buffer, filtered through200 nm filter and applied onto anion-exchange chromatography column onfast protein liquid chromatography instrument (GE Healthcare).Leghemoglobin can be bound to anion-exchange chromatography matrix andeluted using sodium chloride gradient. Purity (partial abundance) ofleghemoglobin can be analyzed by SDS-PAGE and determined to be ˜60-80%.

Undesired small molecules from legume roots can be removed from purifiedleghemoglobin by passing leghemoglobin in solution over anion-exchangeresin. These small molecules imbue varying shades of brown color toroot-root nodule lysates, thus decreasing the color quality ofleghemoglobin solution. In one embodiment, anion-exchange resin is FFQ,DEAF, Amberlite IRA900, Dowex 22, or Dowex 1x4. Leghemoglobin purifiedeither by ammonium sulfate fractionation (60% wt/v and 90% wt/v ammoniumsulfate) or by anion-exchange chromatography was buffer exchanged into20 mM potassium phosphate pH 7.4, 100 mM sodium chloride and solutionpassed over one of the above mentioned anion-exchange resins.Flowthrough can be collected and its colored compared to the color ofthe solution before passage over anion-exchange resins. Colorimprovement to purified leghemoglobin solution as evaluated by visualinspection can be observed (from yellow/brown to more apparent red),however to different extent of removal of yellow-brown tinge.

Alternatively, the heme-containing protein can be recombinantly producedas described in section III B. For example, a non-symbiotic hemoglobinfrom moong bean can be recombinantly expressed in E. coli and purifiedusing anion-exchange chromatography or cation-exchange chromatography. Acell lysate can be loaded over FF-Q resin on fast protein liquidchromatography instrument (GE Healthcare). Moong bean non-symbiotichemoglobin eluted in the flowthrough fractions. Purity (partialabundance) of Moong bean non-symbiotic hemoglobin was analyzed by SDS-PAGE and determined to be as a fraction of total protein: 12% in E.coli lysate, and 31% after purification on FFQ. UV-Vis analysis ofpurified protein showed spectra characteristic of heme bound protein.

Alternatively, the cell lysate can be loaded over a FF-S resin on a fastprotein liquid chromatography instrument (GE Healthcare). Moong beannon-symbiotic hemoglobin can be bound to FF-S column and eluted usingsodium chloride gradient (50 mM-1000 mM). Purity (partial abundance) ofMoong bean non-symbiotic hemoglobin can be analyzed by SDS-PAGE anddetermined to be: E. coli lysate 13%, after purification on FFQ 35%.UV-Vis analysis of purified protein can show spectra characteristic ofheme bound protein.

In some embodiments, the heme proteins are utilized as ingredients infood products where the flavor of blood is desired. Heme-containingproteins of the invention were tasted by a panel of volunteers and ineach case described as tasting like blood.

Heme proteins, for example leghemoglobin, can be combined with otherplant based meat replica components. In some embodiments the hemeproteins are captured in a gel which contains other components, forexample lipids and or other proteins. In some aspects, multiple gels arecombined with non-gel based heme proteins. In some embodiments, thecombination of the heme proteins and the other compounds of theconsumable are done to insure that the heme proteins are able to diffusethrough the consumable. In some embodiments the consumable is soaked ina heme-protein containing solution, for instance a leghemoglobinsolution, e.g., for 1, 5, 10, 15, 30, or 45 minutes or for 1, 5, 10, 15,20 or 30 hours.

Given the usefulness of heme proteins for coloring consumables, it isuseful to detect whether a product contains a particular heme protein.Accordingly the present invention includes in some embodiments methodsto determine whether a product contains a heme protein. For example, anELISA, a proximity-ligation assay, a luminex assay, or western blotanalysis can be performed to determine whether leghemoglobin or otherheme-containing protein is present in a food product such as meat or ameat replica. In one embodiment the detection methods are performed todetermine whether meat has been altered with leghemoglobin or otherheme-containing protein.

E. Mayonnaise Spread Replica.

Mayonnaise is thick, creamy sauce. Traditional mayonnaise is a stableemulsion of oil and egg yolk. It is thought that lecithin and proteinsfrom egg yolk stabilize the emulsion. Traditional commercial mayonnaisetypically contains 70-80% (wt/wt) fat and 5% (wt/wt) of egg yolk. Lowerfat commercial products can contain ˜20% wt/wt fat. The consumable cancomprise a composition that has comparable properties to mayonnaise.

In one embodiment, purified plant proteins can be used as substitute foregg proteins to make stable, creamy protein-fat emulsions whose visualand mouth-feel appearance resemble traditional mayonnaise. Fat (˜20-80%wt/wt) can be from a single source or from multiple sources as describedherein. Non-traditional mayonnaise products can be used for all theculinary applications that traditional mayonnaise is used for. In oneembodiment, vinegar and/or lemon and/or lime juice are added as flavoradditives. In one embodiment, the purified plant proteins are not soyproteins. In one embodiment, the flavor can be modified by addition ofmustard, spices, herbs, and/or pickles.

A mayonnaise replica can comprise a mixture of non-animal proteins. Inone embodiment, a mayonnaise replica is a mixture of 50% (wt/v) ricebran oil and 7% (wt/v) Moong bean 8S protein. In one embodiment, amayonnaise replica is a mixture of 70% (wt/v) sunflower oil or cocoabutter, 2.4% (wt/v) RuBisCo, 0.29% (wt/wt) soybean lecithin, andoptionally 8 μM oleosin.

The mixture can be emulsified, and the stability of the emulsion can becontrolled by modifying the size of the oil-water-protein particles byhigh pressure homogenization or sonication. Oil can be added as liquid.Protein can be added as solution in buffer. Soybean lecithin can beresuspended in water and sonicated prior to mixing with an oil andprotein solution. A resulting oil, protein and lecithin solution can behomogenized, for example, first at 5000 psi and then at 8000 psi, or canbe sonicated at 40% duty cycle for 2 minutes at maximum setting.Thickness, texture, creaminess and visual appearance of resultingproducts are similar to one of traditional mayonnaise. In some instances(e.g. using Moong bean 8S protein and rice bran oil), a product is alight off-white in color.

F. Cream Liquor Replica

Traditionally, cream liquors contain dairy cream and liquor as theirbase. Examples of liquor include whiskey, Irish whiskey, Scotch whiskey,rum, vodka, grappa, or fermented fruits (e.g., cherry liquor), plumbrandy, tequila, or herbal bitters. A cream liquour replica can beproduced by substituting the dairy cream in cream liquor with anon-dairy cream fraction from plant sources. In one embodiment, dairycream in a cream liquor can be substituted by a stable emulsion of plantfats and isolated or purified proteins of a consistency similar to dairycream. In one embodiment, purified plant proteins and/or plant fats canbe from single or multiple sources as described herein. For example, acream liquor can include a sunflower cream fraction, RuBsiCo andwhiskey, and one or more optional flavorings (e.g., vanilla, chocolate,and or coffee).

G. Protein Enriched Alcoholic Beverage

Traditionally, alcoholic beverages contain negligible to low amounts ofprotein. Addition of plant proteins to various alcoholic beverages wouldpositively modify their flavor, mouthfeel, physical state and increasetheir nutritional protein content. In addition, the presence of proteinin various alcoholic beverages used in cocktails would positively modifythe cocktail's flavor, mouthfeel, physical state and increase theirnutritional protein content. Different classes of alcoholic beveragescontain different amounts of alcohols. For example, wine coolers containabout 4-7% alcohol, beer contains about ˜3-10% alcohol, wine containsabout 8-14% v/v alcohol, dessert wines contain about 17-20% alcohol,whiskey contains about ˜40% alcohol, and vodka contains about 35-50%alcohol. In addition, some traditional alcoholic beverages includesugars (for example, Bacardi Razz at 10% wt/v).

Accordingly beverages containing alcohol can be supplemented by additionof purified plant proteins at for example 0.1-5% wt/v and optionallysugar (1-15% wt/v). The sugar can be, for example, cane sugar, brownsugar, sucrose, or glucose. For example, purified Rubisco at 180 mg/mlin 20 mM K-phosphate pH 7.0, 150 mM NaCl can be added to a whiskey.Jameson whiskey supplemented with 5% wt/v Rubsico formed a soft gel,with a consistency similar to traditional Jello shots.

For example, Rubsico, moong bean 8S, and pa Globulin enriched alcoholicbeverages were made by adding purified Rubsico, Moong bean 8S and peaglobulin proteins at final protein concentrations of 0.5%, 1% and 5%wt/v, respectively, to Corona beer, Pinot grigio wine and Jamesonwhiskey. Zein was added at 0.5%, 1% and 5% wt/v to a 60% ethanol, 5%sucrose solution in water.

Pea proteins were extracted from pea flour by resuspending the flour in5%, 20% or 40% ethanol, 5% sucrose solutions in water, followed byincubation for 1 hr at room temperature. Any undissolved solids wereremoved by centrifugation at 5000 g for 10 min. The resultantsupernatant solution was clear in appearance. The 5% ethanol solutionwas particularly useful.

A sensory panel evaluated all protein enriched alcoholic beverages ashaving aromas and flavors different from beverages not-enriched inprotein. In some cases, generated aromas and flavors were judged asneutral, in some cases as more appealing and in some cases as lessappealing than controls. In particular example, addition of both 0.5%wt/v and 1% wt/v of Moong bean 8S protein to Jameson whiskey softenedthe aroma and mouthfeel of Jameson. Addition of 0.5% wt/v of moong beanto Jameson whisky added a slightly creamy flavor to Jameson, with anaroma similar to a traditional White Russian cocktail. Addition of 5%wt/v zein to Jameson whiskey generated aromas and flavor characterizedas moldy beans and raw potato.

In another example where Corona beer was enriched with 0.5% wt/v peaGlobulin, the aroma changed to hoppy and resembled one of Indian PaleAle, and the flavor changed to carrying pea notes. Addition of 0.5% wt/vand 5% wt/v of moong bean 8S protein changed the Corona aroma towardssweet peony flower with an intensified hop aroma. The flavor was neutralin the case of 0.5% wt/v moong bean 8S and carried planty-nutty notes inthe case of 5% wt/v moong bean 8S.

In another example where Pinot Grigio wine was enriched with 1% wt/vmoong bean 8S protein, additional aroma notes of sweet and citrus weredetected, and the flavor changed to that of carrying notes ofpeanut-butter. Addition of 1% wt/v of Pea Globulins modified the aromato that of strong moldy oak and wet leaves. Flavor was modified to carrynotes of mud. Addition of 5% wt/v of Rubisco generated aroma and flavorof wet hay.

A zein enriched 60% ethanol, 5% sucrose solution carried burnt tortillachip aroma notes compared to a corresponding solution without zein.There was no difference in flavor.

Pea proteins enriched 5%, 20% and 40% ethanol, 5% sucrose solutions alldeveloped earthy aroma and flavor compared to protein free controls. Inaddition, flavor of peas was detected and bitter flavor increased athigher alcohol content.

H. Chocolate Spread

Chocolate spread is a chocolate flavored spread whose traditional mainingredients are cocoa powder, dairy milk, plant oil, and sugar.Traditional chocolate spreads are either firm or soft solids at ambientroom temperature and melt at temperatures below that of cocoa butter.The product can be used as a spread on bread, crepes, pancakes, icingfor cakes and cookies, filling for chocolate confectionary, or non-dairychocolate cake filling.

In one embodiment, dairy milk and milk products such as ice cream, whey,cream, yoghurt, sour cream or butter fat are substituted by a non-dairycream fraction made as described herein. In one embodiment, a non-dairycream fraction comes from a single source or multiple sources describedherein. In one embodiment, dairy milk and milk products are substitutedby any non-dairy milk described herein. In one embodiment, dairy milkand milk products are substituted by purified plant proteins describedherein. In one embodiment, dairy milk and milk products are substitutedby soft solid stable emulsions made from single or multiple plant oiland single or multiple purified plant proteins.

I. Other Applications:

In one embodiment, non-dairy plant cream fraction can be used as asubstitute for dairy milk and dairy milk products to make non-dairy milkchocolate bars or non-dairy milk chocolate confectionary.

In one embodiment, non-dairy plant cream fraction and purified plantproteins can be used as substitute for dairy milk and dairy milkproducts to make non-dairy milk chocolate bars or a non-dairy milkchocolate confectionary.

In one embodiment, non-dairy plant cream fraction and plant proteins canbe used to make chocolate mousse. Traditional main chocolate mousseingredients are bittersweet or semisweet chocolate, dairy butter andeggs. In one embodiment, dairy butter can be substituted by a non-dairyplant cream fraction. In one embodiment, dairy butter and eggs can besubstituted by a non-dairy plant cream fraction and a foam stabilizingplant seed storage proteins such as pea albumin.

In one embodiment, a vegan consumable such as pate analog can be made.Vegan pate analog can be made by finely chopping 10 g of fat replica andheating it on a frying pan with finely chopped shallots for 2-3 min.Muscle replica (20 g) made without connective tissue replica fibers canbe chopped into ½-inch cubes and browned in the fat and shallots mix foranother 3-5 min. The mixture can be forced through a sieve untilhomogeneous. The pan, while still warm, can be rinsed with a tablespoonof madeira without allowing it to evaporate fully. The liquid from thepan is added to the homogenized mix, spices (salt, pepper) are added totaste, and the mix is forced through a sieve again. After chilling in arefrigerator (e.g., for 15 minutes), the pate is ready to be served.

In some embodiments, other fat-to-muscle replica ratios are used tocreate leaner or richer pates. For example, pate can contain 0.5-10%,about 5%-40%, about 10%-60%, or about 30-70%, or >70% of an adiposetissue replica.

In some embodiments, a muscle tissue replica with a higher iron contentcan be used for pate to make it a closer imitation of pig or bird liverpate. For example, muscle tissue replica can contain about 1%, about1.5%, about 2%, or >2% of a heme protein.

In some embodiments, muscle tissue replica with lower iron content canbe used for pate to make it closer imitation of bird meat or fish pate.For example, muscle tissue replica can contain about 1%, about 0.5%,about 0.2%, or <0.2% of a heme protein.

In one embodiment, a vegan consumable such as a blood sausage analog canbe made. Vegan blood sausage is made from a blood analog created bymixing solutions of heme protein and purified plant protein. Forexample, 35 ml of a mixed solution of leghemoglobin (120 mg/ml) and peaalbumin (100 mg/ml), which approximates the composition of blood, can becarefully mixed with a slurry of corn flour in salt water (6:5 w/v flourto water ratio). A tablespoon of chopped onion can be fried with 10 g ofchopped adipose tissue replica, mixed with a few raisins and cooled toroom temperature before mixing with blood/flour mix. The mixture can beseasoned to taste (for example, using salt, pepper, parsley and/orcinnamon), loaded into vegetarian sausage casings, and poached innear-boiling water for about 45 min. After cooling, the sausage can beconsumed as is or further cooked, for example smoked, crisped in an ovenor roasted.

In some embodiments, muscle replica can be included in the recipe toimitate meat/blood sausages. In some embodiments, barley, buckwheat,oat, rice, rye, sorghum, wheat or other grains can be used in the bloodsausage. In some embodiments, bread, chestnuts, potato, sweet potato,starch or other fillers can be added to, or substitute for, grains inblood sausage.

EXAMPLES Example 1: Protein Isolation

All steps were carried out at 4° C. or room temperature. Centrifugationsteps were at 8000 g for 20 mins, 4° C. or room temperature. The flouris suspended in a specific buffer, the suspension is centrifuged and thesupernatant is microfiltered through a 0.2 micron PES membrane and thenconcentrated by ultrafiltration on a 3 kDa, 5 kDa, or 10 kDa molecularweight cutoff PES membrane on a Spectrum Labs KrosFlo hollow fibertangential flow filtration system.

Once fractionated, all ammonium sulfate precipitate fractions ofinterest were stored at −20° C. until further use. Prior to their use inexperiments, the precipitates were resuspended in 10 volumes of 50 mM KPhosphate buffer, pH 7.4, +0.5 M NaCl. The suspensions were centrifugedand the supernatants microfiltered through a 0.2 micron PES membrane andthen concentrated by ultrafiltration on a 3 kDa, 5 kDa, or 10 kDamolecular weight cutoff PES membrane on a Spectrum Labs KrosFlo hollowfiber tangential flow filtration system. Protein composition atindividual fractionation steps was monitored by SDS-PAGE and proteinconcentrations were measured by standard UV-Vis methods.

(i) Pea-albumins: Dry green or yellow pea flour was used as a source ofpea albumins. The flour was suspended in 10 volumes of 50 mM sodiumacetate buffer pH 5 and stirred for 1 hr. Soluble protein was separatedfrom un-extracted protein and pea seed debris by either centrifugation(8000 g, 20 minutes) or filtration through a 5 micron filter.Supernatant or filtrate, respectively, was collected. To this crudeprotein extract, solid ammonium sulfate was added to 50% wt/vsaturation. The solution was stirred for 1 hour and then centrifuged. Tothe supernatant from this step, ammonium sulfate was added to bring to90% wt/v saturation. The solution was stirred for 1 hour, and thencentrifuged to collect the pea albumin proteins in the pellet. Thepellet was stored at −20° C. until further use. Protein was recoveredfrom the pellet and prepared for use as described above, with theexception that final buffer can contain 0-500 mM sodium chloride.

In some embodiments, the flour was suspended in 10 volumes of 50 mMNaCl, pH 3.8 and stirred for 1 hour. Soluble protein was separated fromun-extracted protein and pea seed debris by centrifugation (8000 g, 20minutes). The supernatant was collected and filtered through a 0.2micron membrane and concentrated using a 10 Kda cutoff PES membrane.

(ii) Pea-globulins: Dry green pea flour was used to extract pea globulinproteins. The flour was suspended in 10 volumes of 50 mM potassiumphosphate buffer pH 8 and 0.4M sodium chloride and stirred for 1 hr.Soluble protein was separated from pea seed debris by centrifugation.The supernatant was subjected to ammonium sulfate fractionation in twosteps at 50% and 80% saturation. The 80% pellet containing globulins ofinterest was stored at −20° C. until further use. Protein was recoveredfrom the pellet and prepared for use as described above.

iii) Soybean 7S and 11S globulins: Globulins from soybean flour wereisolated by first suspending lowfat/defatted soy flour in 4-15 volumesof 10 (or 20) mM potassium phosphate pH 7.4. The slurry was centrifugedat 8000 rcf for 20 mins or clarified by 5 micron filtration and thesupernatant was collected. The crude protein extract contained both the7S and 11S globulins. The solution then was 0.2 micron filtered andconcentrated using a 10 kDa molecular weight cutoff PES membrane on aSpectrum Labs KrosFlo hollow fiber tangential flow filtration system orby passing over anion-exchange resin prior to use in experiments. The115 globulins were separated from the 7S proteins by isoelectricprecipitation. The pH of the crude protein extract was adjusted to 6.4with dilute HCl, stirred for 30 min-1 hr and then centrifuged to collectthe 11S precipitate and 7S proteins in the supernatant. The 115 fractionwas resuspended with 10 mM Potassium phosphate pH 7.4 and the proteinfractions were microfiltered and concentrated prior to use.

Soybean proteins also can be extracted by suspending the defatted soyflour in 4-15 volumes (e.g., 5 volumes) of 20 mM sodium carbonate, pH 9(or water, pH adjusted to 9 after addition of the flour) or 20 mMpotassium phosphate buffer pH 7.4 and 100 mM sodium chloride to decreaseoff-flavors in the purified protein. The slurry is stirred for one hourand centrifuged at 8000×g for 20 minutes. The extracted proteins areultrafiltered and then processed as above or alternatively, thesupernatant was collected and filtered through a 0.2 micron membrane andconcentrated using a 10 KDa cutoff PES membrane.

(iv) Moong bean 8S globulins: Moong bean flour was used to extract 8Sglobulins by first suspending the flour in 4 volumes of 50 mM KPhosphatebuffer pH 7 (+0.5M NaCl for lab scale purifications). Aftercentrifugation, proteins in the supernatant were fractionated byaddition of ammonium sulfate in 2 steps at 50% and 90% saturationrespectively. The precipitate from the 90% fraction contained the 8Sglobulins and was saved at −20° C. until further use. Protein wasrecovered from the pellet and prepared for use as described above.

Moong bean globulins also can be extracted by suspending the flour in 4volumes of 20 mM sodium carbonate buffer, pH 9 (or water adjusted to pH9 after addition of the moong flour) to reduce off-flavors in thepurified protein fractions. The slurry is centrifuged (or filtered) toremove solids, ultrafiltered and then processed as described above.

(v) Late embryogenesis abundant proteins: Flour (including but notlimited to moong bean and soy flour) was suspended in 20 mM Tris-HCl, pH8.0, 10 mM NaCl, and stirred at room temperature for 1 hour thencentrifuged. Acid (HCl or acetic acid) was added to the supernatant to a5% concentration (v/v), stirred at room temperature then centrifuged.The supernatant was heated to 95° C. for 15 minutes, and thencentrifuged. The supernatant was precipitated by adding Trichoroaceticacid to 25%, centrifuged, then washed with acetone. Heating and acidwash steps can be carried out in the reverse direction as well.

(vi) Pea-Prolamins: Dry green pea flour was suspended in 5× (w/v) 60%ethanol, stirred at room temperature for one hour, then centrifuged(7000 g, 20 min) and the supernatant collected. The ethanol in thesupernatant was evaporated by heating the solution to 85° C. and thencooling to room temperature. Ice-cold acetone was added (1:4 v/v) toprecipitate the proteins. The solution then was centrifuged (4000 g, 20min), and protein recovered as the light-beige colored pellet.

(vii) Zein-Prolamins: Corn protein concentration or flour was suspendedin 5× (w/v) 60% ethanol, stirred at room temperature for one hour, thencentrifuged. Ethanol in supernatant was evaporated with heat, and thenthe solution is centrifuged, and the protein recovered as the pellet.

(viii) RuBisCO was fractionated from alfalfa greens by first grindingleaves with 4 volumes of cold 50 mM potassium phosphate buffer pH 7.4buffer (0.5M NaCl+2 mM DTT+1 mM EDTA) in a blender. The resulting slurrywas centrifuged to remove debris, and the supernatant (crude lysate) wasused in further purification steps. Proteins in the crude lysate werefractionated by addition of ammonium sulfate to 30% (wt/v) saturation.The solution was stirred for 1 hr and then centrifuged. The pellet fromthis step was discarded and additional ammonium sulfate was added to thesupernatant to 50% (wt/v) ammonium sulfate saturation. The solution wascentrifuged again after stirring for 1 hr. The pellet from this stepcontains RuBisCO, and was kept at −20° ° C. until used. Protein wasrecovered from the pellet and prepared for use as described above.

RuBisCO also can be purified by adjusting the crude lysate to 0.1M NaCland applying to an anion exchange resin. The weakly bound proteincontaminants are washed with 50 mM KPhosphate buffer pH 7.4 buffer+0.1MNaCl. RuBisCO was then eluted with high ionic strength buffer (0.5MNaCl).

RuBisCO solutions were decolorized (pH 7-9) by passing over columnspacked with activated carbon. The colorants bound to the column whileRubisco was isolated in the filtrate.

RuBisCO solutions were also alternatively decolorized by incubating thesolution with FPX66 (Dow Chemicals) resin packed in a column (or batchmode). The slurry is incubated for 30 mins and then the liquid isseparated from the resin. The colorants bind to the resin and RuBisCOwas collected in the column flow-through.

In some embodiments, RuBisCO was isolated from spinach leaves by firstgrinding the leaves with 4 volumes of 20 mM potassium Phosphate bufferpH 7.4 buffer+150 mM NaCl+0.5 mM EDTA) in a blender. The resultingslurry was centrifuged to remove debris, and the supernatant (crudelysate) was filtered through a 0.2 micron membrane and concentratedusing a 10 KDa cutoff PES membrane.

In some embodiments, RuBisCO was extracted from alfalfa or wheatgrassjuice powder by mixing the powder with 4 volumes of 20 mM potassiumPhosphate buffer pH 7.4 buffer+150 mM NaCl+0.5 mM EDTA) in a blender.The resulting slurry was centrifuged to remove debris, and thesupernatant (crude lysate) was filtered through a 0.2 micron membraneand concentrated using a 10 KDa cutoff PES membrane.

(ix) Leghemoglobin. Soy root nodules were suspended and lysed in 20 mMpotassium phosphate pH 7.4, 100 mM potassium chloride and 5 mM EDTAusing grinder-blender. During this process leghemoglobin is releasedinto the buffer. Root-nodule lysate containing leghemoglobin was clearedfrom cell debris by filtration through 5 micron filter. In someembodiments, filtration was followed by centrifugation (7000 g, 20 min).Clarified lysate containing leghemoglobin was then filtered through 0.2micron filter and applied onto anion-exchange chromatography column(High Prep Q; High Prep DEAE, GE Healthtcare) on fast protein liquidchromatography instrument (GE Healthcare). Leghemoglobin was collectedin flowthrough fraction and concentrated over 3 kDa molecular weightcutoff PES membrane on a Spectrum Labs KrosFlo hollow fiber tangentialflow filtration system to a desired concentration. Purity (partialabundance) of purified leghemoglobin was analyzed by SDS-PAGE gel: inlysate leghemoglobin is present at 20-40%, while after anion-exchangepurification it is present at 70-80%. In another embodiment, soybeanleghemoglobin flow through from anion-exchange chromatography wasapplied onto size-exclusion chromatography (Sephacryl S-100 HR, GEHealthcare). Soybean leghemoglobin eluted as two fractions correspondingto dimer and monomer species. Purity (partial abundance) ofleghemoglobin was analyzed by SDS-PAGE and determined to be ˜90-100%.Analysis of UV-VIS spectra (250-700 nm) revealed spectral signatureconsistent with heme loaded leghemoglobin.

(x) Non-symbiotic hemoglobin from moong bean was cloned intopJexpress401 vector (DNA2.0) and transformed into E. coli BL21. Cellswere grown in LB media containing soytone instead of tryptone,kanamycin, 0.1 mM ferric chloride and 10 μg/ml 5-aminolevulinic acid.Expression was induced by 0.2 mM IPTG and cells grown at 30° C. for 20hr. E. coli cells expressing moong bean non-symbiotic hemoglobin werecollected and resuspended in 20 mM IVIES buffer pH 6.5, 50 mM NaCL, 1mmM MgCl₂, 1 mM CaCl₂). Add a bit of DNAaseI, and protease inhibitors.Cells were lysed by sonication. Lysate was cleared from cell debris bycentrifugation at 16 000 g for 20 min, followed by filtration over 200nm filter. Cell lysate was then loaded over FF-S resin on fast proteinliquid chromatography instrument (GE Healthcare). Moong beannon-symbiotic hemoglobin bound to FF-S column and was eluted usingsodium chloride gradient (50 mM-1000 mM). Purity (partial abundance) ofmoong bean non-symbiotic hemoglobin was analyzed by SDS-PAGE anddetermined to be: E. coli lysate 13%, after purification on FFQ 35%.UV-Vis analysis of purified protein showed spectra characteristic ofheme bound protein.

(xi) Hemeproteins were synthesized with an N-terminal His6 epitope tagand a TEV cleavage site, cloned into pJexpress401 vector (DNA2.0), andtransformed into E. coli BL21. Transformed cells were grown in LB mediacontaining soytone instead of tryptone, kanamycin, 0.1 mM ferricchloride and 10 μg/ml 5-aminolevulinic acid. Expression was induced by0.2 mM IPTG and cells grown at 30° C. for 20 hr. E. coli cellsexpressing heme proteins were collected and resuspended in 50 mMpotassium phosphate pH 8, 150 mM NaCl, 10 mM imidazole, 1 mM MgCl₂, 1 mMCaCl₂), DNAaseI, and protease inhibitors. Cells were lysed by sonicationand clarified by centrifugation at 9000×g. Lysate was incubated withNiNTA resin (MCLAB), washed with 5 column volumes (CV) of 50 mMpotassium phosphate pH 8, 150 mM NaCl, 10 mM imidazole, and eluted with50 mM potassium phosphate pH 8, 150 mM NaCl, 500 mM imidazole. SDS-PAGEand UV-vis spectra confirmed expected molecular weights and completeheme-loading, respectively.

In some embodiments, transformed cells were grown in seed mediacomprised of 10 g/L glucose monohydrate, 8 g/L Monopotassium Phosphate,2.5 g/L Sensient Amberferm 6400, 2.5 g/L Sensient Tastone 154, 2 g/LDiammonium Phosphate, 1 mL/L Trace Metals Mixture (Teknova 1000× TraceMetals Mixture Cat. No. T1001), 1 g/L Magnesium Sulfate, 0.25 mL 0.1Msolution Ferric Chloride, 0.5 mL/L Sigma. Anti-foam 204, 1 mL/L,Kanamycin Sulfate 1000× solution. 250 mL of media was used in a (4)-1Lbaffeled shakeflasks, innoculated with 0.25 mL each from a single vialof glycerol stock culture. Shakeflasks were grown for 5.5 hours, with250 RPM agitation at 37° C. 40 L of seed media was steam-sterlized in a100 L bioreactor, cooled to 37° C., pH-adjusted to 7.0 and innoculatedwith 800 mL of shakeflask culture once a shakeflask OD of 2.5 wasachieved. Aeration to the bioreactor was supplied at 40 L/m andagitation was 250 RPM. After 2.2 hours of growth, an OD of 2.20 wasreached and 22 L of culture was transferred to the final 4 m³bioreactor. The starting media for the final bioreactor comprised of thefollowing components steamed-in-place: 1775 L deionised water, 21.75 kgMonopotassium Phosphate, 2.175 kg Diammonium Phosphate, 4.35 kg AmmoniumFerric Citrate, 8.7 kg Ammonium Sulfate, 10.875 kg Sensient Amberferm6400, 10.875 kg Sensient Tastone 154. After 30 minutes of steaming, themedia components were cooled to 37° C. and post-sterilization additionswere made: 2.145 L of 0.1M Ferric Chloride solution, 59.32 kg 55% w/wGlucose Monohydrate, 3.9 L of Trace Metals Mixture (Teknova 1000× TraceMetals Mixture Cat. No. T1001), 10.88 L of 200 g/L Diammonium Phosphate,36.14 L 1M Magnesium Sulfate, and 2.175 L Sigma Anti-foam 204, 2.175 LKanamycin Sulfate 1000× solution. pH was controlled at 7.0 via theaddition of 30% Ammonium Hydroxide. Aeration was supplied at 2.175m³/min, dissolved oxygen was controlled at 25% by varying agitationbetween 60-150 RPM. At two timepoints (EFT=4 and EFT8), bolus additionsof additional nutrients were supplied. Each addition added 5.5 kg ofSensient Amberferm 6400, 5.5 kg of Sensient Tastone 154 and 4.4 kg ofDiammonium Phosphate, in autoclaved solutions (100 g/L solution forAmberferm and Tastetone, 200 g/L for Diammonium Phosphate). A sterileglucose solution of 55% w/v Glucose Monohydrate was fed into thebioreactor to maintain a level of residual glucose of 2-5 g/L. Once anOD of 25 was reached, the temperature was reduced to 25° C. and theculture was induced with 0.648 L of 1M Isopropylβ-D-1-thiogalactopyranoside. The culture was allowed to grow for a totaltime of 25 hours, at which point the culture was diluted 1:1 withdeionized water, then centrifuged, concentrating the centrate to 50% v/vsolids content. Cell centrate was frozen at −20° C. Centrate was thawedto 4° C. and diluted in 20 mM potassium phosphate pH 7.8, 100 mM NaCl,10 mM imidazole, and homogenized at 15,000 PSI. Homogenized cells were0.2 urn filtered by tangential flow filtration (TFF) and filtered lysatewas loaded directly onto a zinc-charged IMAC column (GE). Bound proteinswere washed with 10 column volumes (CV) 20 mM potassium phosphate pH7.4, 100 mM NaCl, 5 mM histidine and eluted with 10 CV 500 mM potassiumphosphate monobasic, 100 mM NaCl. Eluted leghemoglobin was concentratedand diafiltered using a 3 kDa molecular weight cutoff PES membrane andTFF. The concentrated sample was reduced with 20 mM sodium dithioniteand desalted using G-20 resin (GE). Desalted leghemoglobin samples werefrozen in liquid nitrogen and stored at −20 C. Leghemoglobinconcentration and purity were determined by SDS-PAGE and UV-visanalysis.

(xi) Oleosin. Sunflower oil bodies were purified from sunflower seeds.Sunflower seeds were blended in 100 mM sodium phosphate buffer pH 7.4,50 mM sodium chloride, 1 mM EDTA at 1:3 wt/v. Oil-bodies were collectedby centrifugation (5000 g, 20 min), and resuspended at 1:5 (wt/v) in 50mM sodium chloride, 2M urea and stir for 30 min, 4° C. 2M urea wash andcentrifugation steps were repeated. Oil-bodies collected bycentrifugation were resuspended in 100 mM sodium phosphate buffer pH7.4, 50 mM sodium chloride. Centrifugation and washing steps wererepeated once more, and the final washed oil-bodies fraction wasobtained from a last centrifugation step. Oil-bodies were resuspended at10% wt/w in 100 mM sodium phosphate buffer pH 7.4, 50 mM sodiumchloride, 2% wt/v vegetable oil fatty acid salts, homogenized at 5000psi and incubated at 4° C. for 12 hr. Solution was centrifuged (8000 g,30 min), top layer removed and soluble fraction collected. SDS-PAGEanalysis suggested that oleosins are a major protein present in thesoluble fraction. Oleosin concentration was 2.8 mg/ml.

(xii) Pea total proteins: Dry green or yellow pea flour was used toextract total pea proteins. The flour was suspended in 10 volumes of 20mM potassium phosphate buffer pH 8 and 100 mM sodium chloride andstirred for 1 hr. Soluble protein was separated from pea seed debris bycentrifugation. The supernatant was collected and filtered through a 0.2micron membrane and concentrated using a 10 Kda cutoff PES membrane.

(xiii) Pea vicilin and Pea legumin: Dry green or yellow pea flour wasused to extract total pea proteins as described above. The crude peamixture obtained thereof was fractionated into pea vicilin and pealegumin using ion-exchange chromatography. Material was loaded on QSepharose FastFlow resin and fractions were collected as saltconcentration was varied from 100 mM to 500 mM NaCl. Pea vicilin wascollected at 350 mM sodium chloride while pea legumin was collected at460 mM sodium chloride. The collected fractions were concentrated usinga 10 KDa cutoff PES membrane.

(xv) Lentil total proteins: Air classified lentil flour was used toextract crude mixture of lentil proteins. Flour was suspended in 5volumes of 20 mM potassium phosphate buffer pH 7.4 and 0.5 M sodiumchloride and stirred for 1 hr. Soluble protein was separated fromun-extracted protein and lentil seed debris by centrifugation (8000 g,20 minutes). The supernatant was collected and filtered through a 0.2micron membrane and concentrated using a 10 KDa cutoff PES membrane.

(xvi) Lentil albumins: Air classified lentil flour was suspended in 5volumes of 50 mM sodium chloride, pH 3.8 and stirred for 1 hr. Solubleprotein was separated from un-extracted protein and lentil seed debrisby centrifugation (8000 g, 20 minutes). The supernatant was collectedand filtered through a 0.2 micron membrane and concentrated using a 10KDa cutoff PES membrane.

(xvii) Chickpea/Garbanzo bean total proteins: Garbanzo bean flour wassuspended in 5 volumes of 20 mM potassium phosphate buffer pH 7.4 and0.5 M sodium chloride and stirred for 1 hr. Soluble protein wasseparated from un-extracted protein and chickpea seed debris bycentrifugation (8000 g, 20 minutes). The supernatant was collected andfiltered through a 0.2 micron membrane and concentrated using a 10 KDacutoff PES membrane.

(xviii) Chickpea/Garbanzo bean albumins: Garbanzo bean flour wassuspended in 5 volumes of 50 mM sodium chloride, pH 3.8 and stirred for1 hr. Soluble protein was separated from un-extracted protein and lentilseed debris by centrifugation (8000 g, 20 minutes). The supernatant wascollected and filtered through a 0.2 micron membrane and concentratedusing a 10 KDa cutoff PES membrane.

(xix) Amaranth flour dehydrins: Amaranth flour was suspended in 5volumes of 0.5 M sodium chloride, pH 4.0 and stirred for 1 hr. Solubleprotein was separated from un-extracted protein and lentil seed debrisby centrifugation (8000 g, 20 minutes). The supernatant was collectedand filtered through a 0.2 micron membrane and concentrated using a 3KDa cutoff PES membrane. Further enrichment of dehydrins from thisfraction was obtained by boiling the concentrated protein material,spinning at 8000 g for 10 minutes and collecting the supernatant.

Example 2: Constructing a Muscle Tissue Analog

To prepare a muscle tissue replica, 8 ml of moong bean protein solution(114 mg/ml in 20 mM phosphate buffer (pH 7.4) and 400 mM sodiumchloride) were mixed with 16 ml of leghemoglobin solution (6 mg/mlleghemoglobin in 20 mM potassium phosphate, 400 mM NaCl, pH 7.3). Theresulting mixture was concentrated using Amicon spin concentrators (10kDa cut-off) to a final concentration of moong bean 8S globulin 61mg/ml, and of leghemoglobin 6.5 mg/ml. Approximately 400 mg oftransglutaminase powder were added to the solution, which werethoroughly mixed, and divided into two 50 ml Falcon tubes and incubatedovernight at room temperature. Final total protein concentrations was67.5 mg/ml total protein. The muscle tissue replica formed an opaque gelof reddish-brown color, with small amounts (<1 ml) of inclusions of darkred, venous blood colored liquid.

Example 3: Increased Tensile Strength Adipose Tissue Replica

A 40 ml aliquot of rice bran oil and a 40 ml aliquot of moong beanprotein (114 mg/ml) were combined in a 250 ml Pyrex beaker. The beakerwas placed in a water bath and emulsified using a Branson Sonifer 450sonicator with a 12 mm tip for a 6 minute, 60% duty cycle at power level5.

In an 18 cm×18 cm×2.5 cm synthetic rubber Ikea plastic ice cube tray, 48mg of electrospun fibers (from connective tissue Example 14) were laidlongitudinally and as homogenously as possible across the bottom of onetriangular 13.97 cm×1.27 cm×1.5875 cm mold. Approximately 20 ml of therice bran oil/moong bean protein emulsions then were poured on top ofthe fibers. An additional 20 ml of the emulsions were then poured into asimilarly sized blank mold on the same tray to be used as a control.

The ice cube tray was floated in boiling water for 15 minutes, removed,and cooled to room temperature.

Using a razor blade, each of the resulting gels was cut into 3 segments,each 4.66 cm long with a cross-sectional area of 1 cm². A Stable MicroSystems TA XTExpress Enhanced texture analyser with attached TA-96Bprobe was used to assess the tensile strength. The fiber containing thefat replica had a tensile strength of 23 kPa, whereas the fat replicawith no fibers had a tensile strength of 20 kPa.

Example 4 High Percentage Fat Adipose-Replica

An adipose tissue replica comprising a protein-oil emulsification formedwith 3.3% wt/v pea globulin, 70% v/v oil that consisted of an equalmixture of coconut, cocoa, olive, and palm oils, and 0.5% wt/v lecitin,was cross-linked with a 2% transglutaminase (Ajinomoto Activa® TI).After draining and dehydrating, the resulting gel was medium soft andthe fat content was confirmed to be 75% (wt/wt).

An adiose tissue matrix comprising a protein-oil emulsification wasformed with 1.6% wt/v Rubisco and 80% v/v cocoa butter. The resultinggel was soft.

Example 5: Method of Preparing Adipose Tissue Replicas

Oils are melted if necessary by warming to room temperature or gentlyheated. If the oils are solid at room temperature, they are kept nearthe melting point during the rest of the procedure. Proteins areobtained per specified protocols (see Example 1). Lecithin is weighedand resuspended in water, then sonicated to create a homogenoussolution. Components are combined at specified ratios and brought tovolume with buffer if necessary (20 mM sodium phosphate pH 7.4 with 50mM sodium chloride) then are subjected to homogenization or sonicationto control particle size within an emulsion. Afterwards, emulsions aregelled by either: (a) heating/cooling, (b) cross-linking with atransglutaminase enzyme or (c) heating/cooling followed by addition of atransglutaminase enzyme. Control samples (no heating/cooling nortransglutaminase cross-linking treatments) were prepared forcomparisons. Emulsions stabilized by heating/cooling treatment areprepared by placing emulsion in 90-100° C. water bath for five minutes,then letting samples slowly cool to room temperature. Emulsionsstabilized by transglutaminase cross-linking are prepared by addingtransglutaminase to 2% wt/v and incubating at 37° C. for 12-18 hr.Emulsions stabilized by heating/cooling followed by addition oftransglutaminase enzyme were prepared by first undergoing the heat/coolprotocol, then adding the enzyme once the samples cooled to roomtemperature. All emulsifications are incubated for 8-12 hours at 37° C.

Example 6: Method to Analyze Adipose Tissue Replicas

After different gelling treatments, gelled emulsions are moved to roomtemperature for evaluation. The total volume of gelled emulsions, andvolumes of phase separated water and/or oil volumes (if gelled emulsionsare not in a single phase) are recorded. Firmness of the adipose tissuereplica is evaluated by gentle poking of gelled emulsions. Cookingexperiments are performed by transferring the mass to a heated surfaceand measuring the temperature of the liquid immediately after cooking.

Example 7: Adipose Replica of Beef Fat

An adipose tissue replica was made by gelling a solution of purifiedmoong bean 8S protein emulsified with equal amounts of cocoa butter,coconut butter, olive oil and palm oil. Moong bean 8S protein waspurified as described in Example 1, and had a concentration of 140 mg/mlin 20 mM K-phosphate pH 7.4, 400 mM NaCl. A fat mixture was prepared bymelting individual fats from solid to liquid state at 45° C. for 30 min.Individual fats (cocoa butter, coconut butter, olive oil and palm oil)in liquid states were then mixed at a 1:1:1:1 (v/v) ratio. A protein-fatemulsion was formed by mixing a 70% v/v liquid fat mixture with 4.2%wt/v moong bean 8S protein, 0.4% wt/v soybean lecithin and emulsified byvortexing for 30 sec followed by sonication for 1 min. Afterhomogenization, the fat-protein emulsion was in a single liquid phase asjudged by visual observation.

One adipose tissue replica emulsion was stabilized by cross-linking with0.2% wt/v transglutaminase enzyme at 37° C. for 12 hr. Another fattissue replica was stabilized by gelling of proteins by heating to 100°C. in a water bath followed by cooling to ambient room temperature. Theresulting adipose tissue replicas were in a single phase. The adiposetissue replica matrix formed by the transglutaminase was a softer solidthan the adipose tissue replica matrix formed by heat/cool inducedgelling.

Example 8: An Adipose Replica of Wagyu Beef Fat

An adipose tissue replica was made by gelling an emulsion of purifiedpea globulin proteins and equal amounts of cocoa butter, coconut butter,olive oil and palm oil. Pea globulin proteins were purified as describedin Example 1 and had a concentration of 100 mg/ml, in 20 mM K-phosphatepH 8, 400 mM NaCl. A fat mixture was prepared by melting individual fatsfrom solid to a liquid state at 45° C. for 30 min. Individual fats(cocoa butter, mango butter, olive oil) in liquid state where then mixedat 2:1:1 (olive oil:cocoa butter:mango butter) v/v ratio. A protein-fatemulsion was formed by mixing a liquid fat mixture with a 5% wt/vsolution of pea globulins in a 1:1 ration, and emulsifying using ahand-held homogenizer at the maximum setting for 30 sec. Afterhomogenization, the fat-protein emulsion was in a single liquid phase asjudged by visual observation. Emulsion was stabilized by cross-linkingwith 0.2% wt/v transglutaminase enzyme at 37° C. for 12 hr. Theresulting adipose tissue replica was in a single phase, was a soft solidand was salty in flavor.

Example 9: Adipose Tissue Replica with Fatty Acid Distribution of Beef

An adipose tissue replica was made by gelling an emulsion of purifiedpea globulin proteins and equal amounts of cocoa butter, mango butter,olive oil and rice bran oil. Pea globulin proteins were purified asdescribed in Example 1, and had a concentration of 100 mg/ml, in 20 mMK-phosphate pH 8, 400 mM NaCl. A fat mixture was prepared by meltingindividual fats from solid to liquid state at 45° C. for 30 min.Individual fats (cocoa butter, mango butter, olive oil and rice branoil) in a liquid state where then mixed at 1:1:1:1 v/v ratio. Aprotein-fat emulsion was formed by mixing 50% v/v liquid fat mixturewith 5% wt/v Pea globulins, and emulsifying using a hand-heldhomogenizer at the maximum setting for 30 sec. After homogenization, thefat-protein emulsion was in a single liquid phase as judged by visualobservation. The emulsion was stabilized by cross-linking with 0.2% wt/vtransglutaminase enzyme at 37° C. for 12 hr. The resulting adiposetissue replica was in a single phase, was a soft solid and was salty inflavor.

Example 10: Adipose Tissue Replica where Firmness of Fat Tissue atRefrigeration and Ambient Temperatures is Controlled by MeltingTemperature of Fat in Adipose Tissue Replica

A adipose tissue replica made as a stable emulsion of RuBisCo withsunflower oil is softer than a adipose tissue replica made as a stableemulsion of RuBisCo and cocoa butter. Adipose tissue replicas wereformed with 0.18%, 1.6%, and 2.4% wt/v Rubisco with 70%, 80%, and 90%v/v sunflower or cocoa butters. Each adipose tissue replica thatcontained cocoa butter was firmer than the corresponding replicas thatwere formed with sunflower oil. Adipose tissue replicas comprising0.18%, 1.6%, and 2.4% wt/v Rubisco with 70%, 80%, and 90% v/v cocoabutter were solid at room temperature but melted at close to mouthtemperature. In the adipose tissue replicas formed with varyingconcentrations of Rubisco (0.18, 1.6 1.9% wt/v) and 70-80% v/v sunfloweroil, the replicas were firmer as the amount of protein in the adiposetissue replica matrix increased. Adipose tissue replicas with 0.18% wt/vRubisco were very soft; adipose tissue replicas with 1.6% wt/v Rubiscowere soft; and adipose tissue replicas with 1.9% wt/v RuBisco were ofmedium firmness.

Adipose tissue replicas made as a stable emulsion of Moong Bean 8Sprotein with sunflower oil were softer than adipose tissue replicas madeas a stable emulsion of Moong Bean 8S protein and cocoa butter. Adiposetissue replicas were formed with 2%, 1%, and 0.5% wt/v Moong Bean 8Sprotein with 70%, 80%, and 90% v/v sunflower or cocoa butters. Eachadipose tissue replica that contained cocoa butter was firmer than thecorresponding replica that was formed with sunflower oil.

Adipose tissue replicas made as stable emulsions of Moong Bean 8Sprotein with canola oil were softer than the corresponding adiposetissue replicas made as stable emulsions of Moong Bean 8S protein withan equal mixture of coconut, cocoa, olive, and palm oils. Adipose tissuereplicas were formed with 1.4% wt/v Moong Bean 8S protein with 50%, 70%,and 90% v/v sunflower or mixture of oils. Each adipose tissue replicathat contained a mixture of oils was firmer than the correspondingreplica that was formed with sunflower oil. A adipose tissue replicacomprising 1.4% wt/v Moong Bean 8S protein with 50%, 70%, and 90% v/v ofan equal mixture of coconut, cocoa, olive, and palm oil was solid atroom temperature but melted at close to mouth temperature.

Adipose tissue replica made as a stable emulsion of soy proteins withsunflower oil was softer than the adipose tissue replica made as stableemulsion of soy proteins and cocoa butter. Adipose tissue replicas wereformed with 0.6%, 1.6%, and 2.6% wt/v Soy with 50%, 70%, 80%, and 90%v/v sunflower or mixture of oils. Each adipose tissue replica thatcontained mixture of oils was firmer than the corresponding replica thatwas formed with sunflower oil. Adipose tissue replicas comprising 0.6%,1.6%, and 2.6% wt/v soy proteins with 50%, 70%, 80%, and 90% v/v cocoabutter were solid at room temperature but melted at close to mouthtemperature.

Example 11: Adipose Tissue Replica Cooking: Structure of Fat TissueMatrix Controls Melting Point During Cooking

An adipose tissue comprising a stabilized protein-oil emulsionconstructed as described in Example 5 and Example 6 above, formed with2% w/v Rubisco and 50%, 70%, and 90% v/v cocoa butter, melted at ahigher temperature when formed upon heat/cool denaturation and at alower temperature than when formed by cross-linking with atransglutaminase.

Example 12: Cooking Adipose Tissue: Arrangement and StructuralOrganization of Proteins and Fat within a Fat Tissue Matrix ControlsAmount of Fat Released and Fat Retained by the Adipose Tissue ReplicaDuring Cooking

During cooking of a adipose tissue replica matrix comprising aprotein-oil emulsion formed with 2% w/v Rubisco and 50%, 70%, or 90% v/vcocoa butter, more adipose tissue replica mass was retained aftercooking when the adipose tissue replica was formed upon heat/cooldenaturation than when formed by cross-linking with a transglutaminase.Released mass was liquid and appeared oily.

During cooking of adipose tissue replica matrix comprising a protein-oilemulsion formed with 2.6 and 0.6% w/v Soybean protein and 50%, 70%, or90% v/v cocoa butter, more adipose tissue replica mass was retained whenformed upon heat/cool denaturation than when formed by cross-linkingwith transglutaminase. Released mass was liquid and appeared oily.

Example 13: Cooking an Adipose Tissue Replica: Concentration ofParticular Proteins within Fat Tissue Matrix Controls Mass of AdiposeTissue Replica that Remains after Cooking

A series of adipose tissue replicas constructed from 1.4% wt/v moongbean 8S protein with 90% v/v canola oil and 0.45% wt/v soybean lecithin,were homogenized and increasing concentration of sunflower oleosins wereadded at varying concentrations to the emulsion. The concentration ofoleosin varied from 1:10 to 1:10⁶ oleosin to triglyceride molar ratio.An increase in mass retention after cooking was observed when the ratioof oleosins to oil in adipose tissue replica was greater.

A series of adipose tissue replicas formed with varying concentrationsof Rubisco with 70% v/v sunflower oil, retained more mass upon cookingas the concentration of Rubisco increased. Adipose tissue replicascontaining RuBisCo at 0% wt/v completely melted, while 1.9% wt/v Rubiscoretained 10% mass, and adipose tissue replica containing 2.4% wt/vRubisco retained 20% mass upon cooking

Example 14: Connective Tissue Analog

Connective tissue fiber replicas were manufactured by electrospinning asolution of moong bean globulin (22.5 mg/ml) containing 400 mM sodiumchloride, 6.75% w/v of poly(vinyl alcohol) and trace amounts of sodiumazide (0.007% w/v). The resulting solution was pumped at 3 μl/min usinga syringe pump, from a 5 ml syringe through a Teflon tube and a blunted21 gauge needle. The needle was connected to a positive terminal of aSpellman CZE 30 kV high voltage supply set at 17 kV and fixed 12 cm froma an aluminum drum (ca. 12 cm long, 5 cm in diameter) that was wrappedin aluminum foil. The drum was attached to a spindle that is rotated byan IKA RW20 motor at about 220 rpm. The spindle was connected to aground terminal of the high voltage supply. The protein/polymer fibersthat accumulated on the foil were scraped off and used as the connectivetissue replicas.

Example 15: Extending the Lifetime of Reduced (Heme-Fe2+) Leghemoglobin

Equine myoglobin was purchased from Sigma. Myoglobin was resuspended at10 mg/ml in 20 mM potassium phosphate, pH 8.0, 100 mM NaCl. SDS-PAGEanalysis suggested that protein purity was ˜90%.

Soy leghemoglobin was purified from Glycine max root nodules viaammonium sulfate precipitation (60%/90% fractionation) as detailed inExample 1. Resuspended 90% ammonium sulfate leghemoglobin was furtherpurified by anion-exchange chromatography (HiTrap Q FF 5 mL FPLC column)in 20 mM potassium phosphate, pH 8.0, 100 mM NaCl. The leghemoglobineluted in flow through fractions. SDS-PAGE analysis suggested thatprotein purity was ˜70%. Leghemoglobin was buffer exchanged into 20 mMpotassium phosphate, pH 7.4, 100 mM NaCl and concentrated to 10 mg/ml on3.5 kDa membrane concentrators.

Carbon monoxide treatment: Myoglobin at 10 mg/ml in 20 mM potassiumphosphate, pH 8.0, 100 mM NaCl and leghemoglobin at 10 mg/ml in 20 mMpotassium phosphate, pH 7.4, 100 mM NaCl were first degassed undervacuum for 1 hour at 4° C. then perfused with carbon monoxide gas for 2minutes. Globins were then reduced from heme-Fe³⁺ to heme-Fe²⁺ state byadding 10 mM sodium dithionite, 0.1 mM sodium hydroxide for 2 minutes.Sodium dithionite and sodium hydroxide were removed from the proteinsolution by using size-exclusion chromatography (PD-10 desalting column)in 20 mM potassium phosphate, pH 8.0, 100 mM NaCl and 20 mM potassiumphosphate, pH 7.4, 100 mM NaCl respectively. Globins fractions werecollected as a peak red colored fractions as evaluated by visualestimation. UV-VIS spectra confirmed the presence of heme-Fe²⁺ state forboth proteins. After desalting, the solution was again perfused with gasfor another 2 minutes. The color of the solutions was evaluated bytaking UV-Vis spectra (250 nm-700 nm) every 20 minutes using thenanodrop spectrophotometer. Control samples were not treated with carbonmonoxide.

Sodium nitrite treatment: Myoglobin at 10 mg/ml in 20 mM potassiumphosphate, pH 8.0, 100 mM NaCl and leghemoglobin at 10 mg/ml in 20 mMpotassium phosphate, pH 7.4, 100 mM NaCl were reduced from heme-Fe³⁺ toheme —Fe²⁺ by adding 10 mM sodium dithionite, 0.1 mM sodium hydroxidefor 2 minutes. Sodium dithionite and sodium hydroxide were removed fromthe protein solution by using size-exclusion chromatography (PD-10desalting column) in 20 mM potassium phosphate, pH 8.0, 100 mM NaCl and20 mM potassium phosphate, pH 7.4, 100 mM NaCl, respectively. Globinsfractions were collected as peak red colored fractions as evaluated byvisual estimation. UV-VIS spectra confirmed the presence of heme-Fe²⁺state for both proteins. Sodium nitrite was then added to a finalconcentration of 1 mM from 100 mM nitrite in phosphate buffer pH 7.4.The lifetime of heme-Fe²⁺ state was followed by recording UV-VIS spectra(250-700 nm) using spectrophotometer as a function of time. Controlsamples were not treated with sodium nitrite.

Data analysis of heme-Fe²⁺ life-times for myoglobin and leghemoglobinsamples treated with carbon monoxide and sodium nitrite was performed inMicrosoft Excel by plotting the amplitude of the absorbance peak at the540 nm wavelength. The “baseline” of the 540 nm absorbance wasdetermined by the state of the UV-vis spectrum of the globin solutionsprior to the addition of any additives, dithionite reduction, ordesalting. The built-in curve fit function was used to produce anexponential line-of-best-fit, the exponent of which directly relates tothe half life of the peak amplitude.

The life-time of the heme-Fe²⁺ state and accompanying red color ofmyoglobin and leghemoglobin solutions in the absence of carbon monoxideand sodium nitrite were ˜6 hr and ˜4 hr respectively. Addition of sodiumnitrite extended the life-time of the heme-Fe²⁺ state and accompanyingred color to more than seven days. Addition of carbon monoxide extendedthe life-time of heme-Fe²⁺ state and accompanying red color to more thantwo weeks.

Example 16: Preparation of Meat Replicas in which the Particle Size ofIndividual Tissue Replica Units is Varied to Control Aroma GenerationDuring Cooking

Muscle tissue replica and adipose tissue replica were preparedseparately and then combined into a meat tissue replica such that thesize of individual tissue replica units was varied to control aromageneration during cooking. Individual fat, muscle and connective tissuereplicas were constructed in the following manner.

A muscle tissue replica was prepared as in Example 2. The muscle tissuereplica formed an opaque gel of reddish-brown color, with small amounts(<1 ml) of inclusions of dark red, venous blood colored liquid. Aconnective tissue replica was prepared as in Example 14. An adiposetissue replica was prepared as in Example 7.

Meat replicas with a lean-to-fat ratio 85/15 were prepared by combiningindividual muscle, connective and adipose tissues such that particlesize of individual tissues replicas varied. (a) 2.1 g of muscle replicawith 0.9 g of chunks of fat replica 5-10 mm in size (“coarse mix”); (b)2.1 g of muscle replica with 0.9 g of fat replica chopped to 2-3 mm insize (“fine mix”); and (c) 2.1 g of muscle replica with 0.9 g of fatreplica thoroughly blended to <1 mm in size (“blend”). “Muscle only”control sample contained 3 g of muscle replica alone. “Fat only” controlsample contained 3 g of fat replica alone as 5-10 mm sized particle.Meat, muscle and fat tissue samples were cooked in sealed glass vials at150° C. for 10 min. Aroma profiles of the samples were analyzed by apanel of testers, and by GC-MS.

Sensory olfactory analysis of meat replica samples performed by a panelof testers, suggested that the size of individual tissue units and theextent of their mixing within meat tissue replicas correlated withgeneration of different aromas. Muscle tissue replica cooked by itselfgenerated aromas associated with store-bought gravy, faint citrus andstar anise. Adipose tissue replica cooked by itself generated aromasassociated with musty, rancid and sweet aromas. Cooked meat tissuereplica (coarse particle size) generated aromas of store-bought gravy,sweet, slightly musty and star-anise. Cooked meat tissue replica (fineparticle size) generated aromas associated with soy sauce, musty,slightly rancid and beef bouillon. Cooked meat tissue replica (very fineparticle size) generated aromas associated with sweet, musty andsoy-sauce. All samples with the exception of adipose tissue replicagenerated aromas associated with burnt meat smell, however to varyingintensities.

Analysis of GCMS data indicated that the size of individual tissue unitsand the extent of their mixing within meat tissue replicas had profoundeffects on the generation of aromatic compounds upon cooking. Inparticular, multiple aromatic compounds associated with fruity/greanbean/metallic (2-pentyl-furan); nutty/green (4-methylthiazole); peanutbutter/musty (pyrazine, ethyl); raw potato/roasted/earthy (Pyrazine,2,3-dimethyl); vinegary (acetic acid); spicy/caramel/almond(5-methyl-2-furancarboxaldehyde); creamy (butyrolactone); sweet(2,5-dimethyl-3-(3-methyl butyl) pyrazine); fruity/stale beer(2-cyclopentene-1-one, 2-hydroxy-3-methyl);musty/nutty/coumarin/licorice/walnut/bread (3-acetyl-1H-pyrolline);coconut/woody/sweet (pantolactone); penetrating(1-H-pyrrole-2-2carboxaldehyde, 1-methyl); minty (caprolactam); toastycaramel (4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl) aromasappeared only in mixed meat replicas, but not in individual tissuereplicas. Some other aromatic compounds, for example associated withgasoline-like (nonane, 2,6-dimethyl), petroleum-like (3-hexene,3-methyl); sour/putrid/fish-like (pyridine); bland/woody/yogurt(acetoin); fatty/honey/citrus (octanal); pungent/sweet/caramelic(2-propanone, 1-hydroxy) and nutty/burnt green (ethenyl pyrazine) aromasappeared only in individual tissue replicas, but did not accumulate inmixed meat replicas. Furthermore, the levels to which all of the abovecompounds accumulated during cooking depended on the sizes of tissueunits and how they are mixed (coarse particle size, fine particle size,or very fine particle size (blended)).

Similar to meat tissue replica, it was found that structuralorganization and particle size of beef tissues modify response of beeftissues to cooking. For example, the flavor of meat is modified by thesize of the particles. Beef samples were prepared as following: samplesof beef muscle and beef fat were cut separately with a knife and: (a)“ground”, where knife-cut tissue cubes were passed through standard meatgrinder. 80/20 (wt/wt) lean/fat ground beef sample was prepared bymixing muscle and fat tissue cubes at appropriate ratio prior togrinding. This sample preparation is referred to as a “fine sizeparticle mix”. (b) Ground tissue particle size was further reduced byfreezing ground tissue in liquid nitrogen and crushing it using mortarand pestle to a very fine powder (particle size <1 mm). This samplepreparation is referred to as a “very fine size particle mix”. Allsamples were cooked in sealed glass vials at 150° C. for 10 min. Aromaprofiles of the samples were analyzed by a panel of testers, and byGC-MS, as described in Example 1. “Muscle only” control sample contained3 g of muscle tissue alone. “Fat only” control sample contained 3 g offat tissue alone. Ground beef sample contained 3 g of a 80/20 (wt/wt)muscle/fat mixture.

Sensory olfactory analysis of beef samples performed by a panel oftesters, suggested that the size of individual tissue units and theextent of their mixing within the samples correlated with generation ofdifferent aromas. Beef muscle cooked by itself generated typical aromasassociated with cooked ground beef. Fat tissue replica cooked by itselfgenerated slightly sweet aromas, and aromas associated with burntmushrooms. Cooked ground beef with “fine size particle mix” generatedtypical aromas associated with cooked ground beef, with presence ofslightly sweet aromas characteristic of cooked fat. Cooked ground beefwith “very fine size particle mix” generated aromas associated withcooked ground beef, but no slightly sweet aroma characteristic of cookedfat was detected.

Analysis of GCMS data indicated that the particle size of the individualtissue units has effect on generation of aromatic compounds uponcooking. In particular generation and/or amount of multiple aromaticcompounds by individual tissue samples or ground beef sample varied incorrelation with particle size of the tissue. Some of the aromaticcompounds that differed between fine and very fine particle size ofmuscle tissue: 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl,3-Acetyl-1H-pyrolline, 1-(6-methyl-2-pyrazinyl)-1-ethanone,2,5-dimethyl-3-(3-methyl butyl) pyrazine, 2-furancarboxyaldehyde,5-methyl, Acetic acid, Ethenyl pyrazine, Pyrazine, 2,3-dimethyl,2-Propanone, 1-hydroxy, Octanal, Acetoin, 4-Methylthiazole,Pseudo-2-pentyl-furan, 2-pentyl-furan. Some of the aromatic compoundsthat differed between fine and very fine particle size of fat tissue:triethylene glycol: 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl,Caprolactam, 1-(6-methyl-2-pyrazinyl)-1-ethanone, 2-Cyclopentene-1-one,2-hydroxy-3-methyl, Butyrolactone, 2-furancarboxyaldehyde, 5-methyl,Ethanone, 1(2 furanyl), Acetic acid, 2-ethyl-5-methyl pyrazine,Pyrazine, 2,3-dimethyl, Pyrazine, ethyl, Octanal, Acetoin,4-Methylthiazole, Pseudo-2-pentyl-furan, Pyridine, Nonane, 2,6-dimethyl.Some of the aromatic compounds that differed between fine and very fineparticle size of 80/20 muscle/fat sample: 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl, Caprolactam, 1H-1Pyridine,3-carbonitrile, 4-ethyl-2-oxo-2,5, 1-H-Pyrrole-2-2carboxaldehyde,1-methyl, 2-Cyclopentene-1-one, 2-hydroxy-3-methyl,2,5-dimethyl-3-(3-methyl butyl) pyrazine, Butyrolactone,2-furancarboxyaldehyde, 5-methyl, Ethanone, 1(2 furanyl), Acetic acid,Ethenyl pyrazine, 2-ethyl-5-methyl pyrazine, Pyrazine, 2,3-dimethyl;2-Propanone, 1-hydroxy, Octanal, Acetoin, 2-pentyl-furan.

Example 17: Leghemoglobin Contribution to Flavor

Beef flavors and aromas can be created in non beef consumables byaddition of heme proteins. Ground chicken (90% lean, 10% fat) wasstrained with cheesecloth and mixed with recombinant soy leghemoglobinor recombinant bovine myoglobin to a final concentration of 0.5-1.0%wt/wt. The recombinant heme proteins were expressed in E. coli andpurified by nickel affinity purification as described in Example 1.Prior to being mixed with chicken, the heme proteins were reduced with20 mM Na Dithionite. Na dithionite was removed from the sample with aZeba desalting column (Thermo Scientific). Leghemoglobin was desaltedinto 20 mM potassium phosphate pH 7.4, 100 mM NaCl. Myoglobin wasdesalted into either 20 mM potassium phosphate pH 7.4, 100 mM NaCl or 20mM Na citrate pH 6.0, 100 mM NaCl. The reduced heme protein samples weredivided in two, and half the sample was bubbled with carbon monoxide(CO) for 2 minutes. After mixing the heme protein samples with groundchicken, the mixture was poured into nugget-shaped molds and incubatedovernight at 4° C. The nuggets were oven baked or pan fried at 165° C.until each nugget reached an internal temperature of 165° C. A panel ofjudges tasted nuggets containing chicken alone, chicken mixed withbuffer, chicken mixed with either leghemoglobin or myoglobin+/−CO, orbeef (90% lean, 10% fat). Judges filled out a survey to evaluate thearoma and flavor of each nugget. Judges rated the aroma and flavor ofeach nugget as follows: 1=chicken, 2=chicken+faint beef, 3=50/50chicken+beef, 4=beef+faint chicken, 5=beef. Shown in Table 2 are theaverage scores received for each nugget. Percentages indicate the finalconcentration of heme protein wt/wt (abbreviations: KP=20 mM potassiumphosphate pH 7.4, 100 mM NaCl buffer. NC=20 mM Na citrate pH 6.0, 100 mMNaCl buffer. n/d=not determined). Adding recombinant leghemoglobin ormyoglobin to chicken resulted in an increased beef aroma and flavor. Theperceived levels of beef flavor and aroma increased with the myoglobinand leghemoglobin content. Leghemoglobin and myoglobin provide the samebenefit to the flavor and aroma.

TABLE 2 Oven Baked Pan Fried Aroma Flavor Aroma Flavor Chicken 1 1 1 1Chicken KP 1 1 2.5 1.2 Chicken NC 1.5 1.5 1.5 1 Chicken 0.5% legH KP 1.52.5 3.67 3.2 Chicken 0.5% legH + CO KP 2.5 2.5 2.67 2.2 Chicken 0.5% MyoNC 2 2 1.5 2.4 Chicken 0.5% Myo + CO NC 2 2 2.5 3 Chicken 0.5% Myo + COKP 2.5 2.5 2.33 2 Chicken 0.8% Myo + CO NC 2 3 4 2.6 Chicken 1% Myo NC4.5 4 n/d n/d Chicken 1% legH NC 4 4 n/d n/d Beef 5 5 5 5

Example 18: Preparation of a Non-Dairy Cream Liquor

A cream liquor was made from a sunflower cream fraction, RuBsiCo andwhiskey (Jameson). A sunflower cream fraction was made by blendingsunflower seeds in 40 mM sodium phosphate pH 8.0, 400 mM sodium chloridebuffer. Seed debris was pelleted by centrifugation at 5000 g for 20 min,and the cream fraction collected. The cream fraction was resuspended in10 mM potassium phosphate pH 7.4 buffer and collected by centrifugationat 5000 g, 20 min. Rubsico was purified as described in Example 1 andused as 25 mg/ml stock solution in 20 mM K-phosphate pH 7.0, 150 mMNaCl.

In one example, cream liquor was made as follows: 11.4% wt/v ofsunflower cream fraction, 40% v/v Jameson whiskey, 0.4-1.6% wt/vRubsico, 0.5% wt/v vanilla extract, 0.5% v/v espresso coffee, and 1.5%wt/v chocolate powder. The resulting mixture was homogenized at 5000psi.

In another example, cream liquor was made from sunflower cream fractionand whiskey (Jameson) and sugar: 11.4% wt/v of sunflower cream fraction,40% v/v Jameson whiskey, 0.5% v/v vanilla extract, 0.5% v/v espressocoffee, 1.5% wt/v chocolate powder and 8% wt/v sugar.

Beverages were served either at ambient room temperature or chilled.Resulting beverages were beige to light chocolate in color. The tastingresults suggested that the beverage had a very creamy alcoholic flavorsimilar to dairy cream liquor. Chilled product was preferred. Theemulsion was stable at room temperature for at least 1 week (maximumtime tested).

Example 19—Chocolate Spread

A chocolate spread was made from 34% (wt/wt) cane sugar, 22% (wt/wt)cocoa powder, 19% (wt/v) pistachio cream fraction, and 12% (v/v) almondmilk. Cane sugar and cocoa powder (Ghirardelli) were commerciallypurchased. Almond skim milk was made in a following manner: Almonds wereblanched by immersion into 100° C. water for 30 sec. Blanched nuts wererecovered and cooled by immersion into ice-cold water. Almonds were airdried. Almonds were then rehydrated by immersion in 2° C. water for 16hr. Rehydrated almonds were drained, mixed with water at 1:2 wt/v ratioand blended in a Vitamix blender for 5 min. The blended slurry wascollected in chilled container and stirred with a frozen cooling stickto cool. Once the slurry cooled to 10° C., the slurry was placed at 2°C. for up to 12 hours. Almond skim and cream were separated bycentrifugation at 7480 g for 30 min at 4° C. Almond milk separated into3 layers, a dense pellet of insoluble solids, a clear to translucentaqueous layer (which is referred to as the “almond skim milk”), and alighter, creamy, opaque layer (which is referred to as the “almondcream”). Almond milk was then was pasteurized at 75° C. for 16 seconds,chilled and stored at 2° C.

A pistachio cream fraction was prepared by blending pistachios in 100 mMsodium carbonate pH 9.5 buffer with 400 mM sodium chloride and 1 mMEDTA, then centrifuging 5000×g for 20 min. The cream fraction wascollected and washed into the same buffer once more. Aftercentrifugation at 5000 g for 20 min, the cream fraction was collectedand washed into 20 mM sodium phosphate pH 7.4 buffer with 50 mM sodiumchloride and 1 mM EDTA. After centrifugation at 5000 g for 20 min, thecream fraction was collected an washed once more in the neutral (pH 7.4)buffer, centrifuged at 5000 g for 20 min. Pistachio cream fraction wascollected and stored at 4° C.

Chocolate spread was made in the following way. Cane sugar was melted inalmond milk, cocoa powder was added to sugar-milk mixture with stirringand melted. Sugar, milk and cocoa were then added to the pistachio creamfraction and whisked together. Resulting mixture was then poured intomolds and let sit for 24 hours at refrigeration and freezertemperatures.

In another example, chocolate spread was made from 42% (wt/wt) canesugar, 27% (wt/wt) cocoa powder, 31% (wt/v) sunflower cream fraction,and 23% (v/v) almond skim milk. All ingredients and procedures apartfrom sunflower cream fraction were as described above.

The sunflower cream fraction was created from blending sunflower seedsin 5 times the weight to volume of a solution of 40 mM potassiumphosphate pH 8, with 400 mM NaCl, 1 mM EDTA, then cooled to 20° C., andthe slurry was then centrifuged. The top cream layer was removed and isblended in the same buffer, followed by heating for 1 hour at 40° C. Theslurry is cooled down to 20° C. then centrifuged; the cream layer isremoved and mixed with 5 times the weight to volume of 100 mM sodiumcarbonate pH 10 with 400 mM NaCl, then centrifuge. The top layer is thenmixed with 5 times the weight to volume of water and centrifuged again,the resulting cream fraction is very creamy, white, and neutral tasting.

In another example, a chocolate spread was made from 37% (wt/wt) canesugar, 23% (wt/wt) cocoa powder, 13% (wt/v) sunflower cream fraction,and 7% (wt/wt) cocoa butter, and 20% v/v almond skim milk.

In another example, a chocolate spread was made from 37% (wt/wt) canesugar, 23% (wt/wt) cocoa powder, 13% (wt/v) sunflower cream fraction,and 7% (wt/wt) coconut oil 20% v/v almond skim milk.

In another example a chocolate spread was made from 37% (wt/wt) canesugar, 23% (wt/wt) cocoa powder, 13% (wt/v) sunflower cream fraction,and 7% (wt/wt) palm oil, and 20% v/v almond skim milk.

In another example a chocolate spread was made from 1.8% (wt/wt) canesugar, 1.13% (wt/wt) cocoa powder, 88% (wt/v) pistachio cream fraction,and 9% almond skim milk, by whisking equal amounts of spread describedabove and pistachio oil bodies.

In another example, a chocolate spread was made from 8.5% (wt/wt) canesugar, 5.4% (wt/wt) cocoa powder, 81% (wt/v) sunflower cream fraction,and 4.6% (v/v) almond skim milk, by mixing chocolate spread describedabove with sunflower cream at ratio 2:1.

Visual and textural inspection of all products suggested that theyformed stable, solid, creamy spreads at room temperatures. All productswere firm solids at refrigeration and freezer temperatures. Tastingresults of all products suggested very pleasant, rich creamy texturewith product melting in the mouth reviewed positively by tasters.Individual tasters preferences varied with respect to like or dislike ofpistachio flavor, coconut flavor, preference for more or less sweetproduct and more or less cocoa flavor. One particular sample wasdescribed as being similar to a milk chocolate spread, as the sunflowercream fraction contributed a neutral flavor.

Example 20—Generation of Adipose Tissue Replica

Adipose tissue replicas were generated using the ingredients recited inTable 3.

TABLE 3 Adipose Tissue Replica Ingredient % Coconut oil 65 Pea vicillinprotein in buffer 21.3 Cocoa butter 10 Buffer 2.7 Lecithin slurry, 50mg/ml 1 Total 100

Lecithin (SOLEC™ F Deoiled Soy Lecithin, The Solae Company, St. Louis,Mo.) was prepared at a concentration of 50 mg/ml in 20 mM potassiumphosphate, 100 mM NaCl, pH 8.0 buffer and sonicated (Sonifier AnalogCell Disruptor model 102C, BRANSON Ultrasonics Corporation, Danbury,Conn.) for 30 seconds.

The pea vicilin protein was supplied as a liquid containingapproximately 140 mg/g pea vicilin in 20 mM potassium phosphate, 100 mMNaCl, pH 8.0 buffer.

Coconut oil (Shay and Company, Milwaukie, Oreg.) and cocoa butter (CocoaFamily, Duarte, Calif.) were melted by heating to 50-70° C. and thencombined and held warm until needed.

The buffered protein solution, additional buffer, and lecithin slurrywere mixed in a 32 ounce sized metal beaker and equilibrated to roomtemperature. An emulsion was formed using a hand held homogenizer (OMNImodel GLH fitted with G20-195ST 20 mm generator probe, OMNIInternational, Kennesaw, Ga.). The homogenizer probe was put into theprotein lecithin mixture and turned on to speed 4. The warmed oil wasthen added slowly over the course of about 2 minutes while continuouslymoving the probe around in the mixture.

The emulsion was then heat set by placing the metal beaker into a 95° C.waterbath. Using a clean spatula, the emulsion was stirred every 20seconds for 3 minutes total. The beaker was then removed from thewaterbath and stored at 4° C. for several hours until thoroughly cooled.

Example 21—Generation of Raw Tissue Replica

A raw tissue replica was generated using the ingredients recited inTable 4.

TABLE 4 Raw Tissue Replica Ingredient % Buffer 41.6 Heme protein inbuffer 26.7 Pea legumin in buffer, dried 12.1 Pea vicillin in buffer,dried 9.4 Flavor precursor mix, 17x 6.2 Transglutaminase preparation 4Total 100

The buffer was 20 mM potassium phosphate, 100 mM NaCl, pH 7.4. The hemeprotein was prepared at a concentration of 55 mg/g in 20 mM potassiumphosphate, 100 mM NaCl, pH 7.4 buffer. The 17× flavor precursor mixprecursor is described in Example 27. The pea legumin was prepared in 20mM potassium phosphate, 500 mM NaCl, pH 8 buffer and then freeze driedprior to use. The final protein concentration of the dried material was746 mg/g. The pea vicilin was prepared in 20 mM potassium phosphate, 200mM NaCl, pH 8 buffer and then freeze dried prior to use. The finalprotein concentration of the dried material was 497 mg/g.

The liquid ingredients (buffer, heme, and flavor precursor mix) weremixed in a plastic beaker. The dry pea legumin and pea vicilin were thenadded and allowed to fully rehydrate while gently stirring for 1 hour atroom temperature. The dry transglutaminase preparation (ACTIVA® TI,Ajinomoto, Fort Lee, N.J.) was then added and stirred for about 5minutes until dissolved. The stirring was then turned off and themixture was allowed to gel at room temperature until firm. After the gelhad formed the raw tissue replica was stored refrigerated until used.

Example 22—Hard Connective Tissue Replica

A hard connective tissue replica was made as following using soy proteinisolate (Supro Ex38, Solae), wheat gluten (Cargill), and bamboo fiber(Alpha-Fiber B-200, The Ingredient House). The purified proteins werefreeze-dried and milled using a standard coffee grinder. Commerciallyavailable powders of soy protein isolate and wheat gluten were used asreceived.

The connective tissue replica contained 49% soy protein isolate, 49%wheat gluten and 2% bamboo fiber. The ingredients were thoroughly mixedand loaded into the loading tube of the extruder's batch feeder. Atwin-screw extruder (Nano 16, Leistritz Extrusion Corp.), with ahigh-pressure water injection pump (Eldex) and custom-made die nozzles(stainless steel tube, 3 mm ID, 15 cm length, pressure rating 3000+ PSI)attached using a Hy-Lok two-ferule tube fitting and a custom-made diewith a threaded nozzle and 10 mm ID, 20 mm long flow channel was used.

The dry mixture was fed into the extruder at the rate 1 g/min. Water wasfed by the pump into the second zone of the extruder's barrel. The rateof water feeding is adjusted to the rate of dry mixture feeding such asto provide 55% moisture level in the final extrudiate. A temperaturegradient was maintained along the extruder barrel as follows: feedzone—25° C., zone 1—30° C., zone 2—60° C., zone 3—130° C., zone 4—130°C. The die plate was neither actively heated, nor cooled. The die nozzlewas actively cooled (by applying moist tissue replica) to maintainextrudate temperature below 100° C.

Hard connective tissue replica obtained by this process was darkoff-white (“cappuccino”) colored material shaped into 3 mm thickfilaments that had tensile strength similar to animal connective tissue(3 MPa).

Example 23. Soft Connective Tissue Replica

A soft connective tissue replica was made as in Example 22, except thatthe rate of water feeding was adjusted to the rate of dry mixturefeeding to provide 60% moisture level in the final extrudate. Atemperature gradient was maintained along the extruder barrel asfollows: feed zone—25° C., zone 1—30° C., zone 2—60° C., zone 3—115° C.,zone 4—115° C. The die plate was neither actively heated, nor cooled.The die nozzle was actively cooled (by applying moist tissue) tomaintain extrudate temperature below 100° C.

Soft connective tissue replica obtained by this process was lightoff-white colored material shaped into 3 mm thick filaments that had lowtensile strength (<0.1 MPa) and had a significant propensity to splitlongitudinally into bands and thin fibers

Example 24. Thin Connective Tissue Replica (Zein Fibers) Process

A thin connective tissue replica was prepared using zein protein powder,glycerol (FCC grade), polyethylene glycol (PEG 400 or PEG3350), ethanol,sodium hydroxide (FCC grade), and water. The zein powder and PEG3350 ata 35% w/w ratio to zein were dissolved in 85% aqueous ethanol to reach afinal zein concentration of 57% w/w. The pH of the solution was adjustedto 7.0 with 1M solution of sodium hydroxide in ethanol. A syringe pumpwith A 1-12 ml syringe, spinning nozzle (hypodermic needle, 18-27 gauge,or a plastic nozzle, 18-24 gauge), heating silicon tape, and a heatingfan was used, with a collector assembled using a computer-controlledmotor rotating a Delrin rod, which serves as a collector.

This solution was loaded into the syringe, which was mounted onto thesyringe pump, with an 18 gauge plastic tip attached. A silicon heatingtape was looped around the tip to maintain it at elevated temperature.After solution was extruded out of the tip and forms a drop, it waspicked up with a spatula and carefully transferred towards the collectorrod to form a filament between the tip and the collector. The extrusionrate was optimized to produce even flow of material from the tip (5 ml/hfor a 12 ml syringe and 18 gauge tip). The collector rotation speed was3 RPM. The heating fan was positioned to blow hot air onto the spoolingfiber. After spooling, the fibers were cured in a 120° C. oven for 24hours.

Thin connective tissue replica obtained by this process was semi-clearyellow colored material shaped into 300-micrometer thick fibers thatwere semi-flexible in air, and became very flexible and elastic inpresence of water, maintaining high tensile strength similar to animalconnective tissue (6 MPa)

Example 25. Noodles

Noodles were prepared using purified pea vicilin (freeze-dried), and soyprotein isolate (Supro EX38 by Solae, Solbar Q842 (CHS)) or soy proteinconcentrate (Hisolate, Harvest Innovations). To prepare the noodles, 67%soy protein concentrate or isolate, and 33% ground pea vicilin powderwere thoroughly mixed and loaded into the loading tube of the extruder'sbatch feeder. The dry mixture was fed into the extruder at the rate inthe range of 1-2 g/min. Water was fed by the pump into the second zoneof the extruder's barrel at the rate 3.6-5.3 ml/min such as to maintainthe final moisture content in the extrudate at 72.5%. A temperaturegradient was maintained along the extruder barrel as follows: feedzone—25° C., zone 1—30° C., zone 2—60° C., zone 3—100° C., zone 4—100°C. Zone 1 temperature can be varied in the range of 25—45° C. Zone 2temperature can be varied in the range of 45—65° C. Zone 3 and 4temperatures can be varied in the range of 95—100° C. The die plate wasneither actively heated, nor cooled. The die nozzle was passively cooledby ambient air ensuring extrudate temperature below 100° C.

Noodles obtained by this process were light yellow colored materialshaped into 1.5 mm thick filaments that had low tensile strength (<0.1MPa) and a moderately sticky texture.

Example 26. Sticky Tissue Replica Preparation

Sticky tissue replica was prepared using purified pea vicilin(freeze-dried) and purified pea legumin (freeze-dried). To prepare thesticky tissue replica, 50% ground pea vicilin powder and 50% ground pealegumin powder were thoroughly mixed and loaded into the loading tube ofthe extruder's batch feeder. The dry mixture was fed into the extruderat the rate in the range 0.4-0.8 g/min. Water was fed by the pump intothe second zone of the extruder's barrel at the rate in the range1.6-3.2 ml/min, such that the final moisture content of the extrudatewas maintained at 80%. As the total throughput increased from 2 g/min to4 g/min, the screw speed was increased from 100 to 200 RPM. Larger diediameters (4 mm and above) are also helpful for preventing backflow athigher throughputs. A temperature gradient was maintained along theextruder barrel as follows: feed zone—25° C., zone 1—30° C., zone 2—60°C., zone 3—90° C., zone 4—90° C. The die plate was neither activelyheated, nor cooled. The die nozzle was passively cooled by ambient air.The die nozzle is kept free of obstructions by solidifying gel material.

Sticky tissue replica obtained by this process was semi-transparentwater-white colored material shaped into irregular 1-5 cm sized bulbsthat had sticky paste-like texture.

Example 27. Preparation of the Flavor Precursor Mix

A flavor precursor mix was prepared by mixing concentrated stocksolutions of each additive to make a 17× solution. Table 5 contains thechemical composition of the mix and the mM concentration of eachcomponent in the final burger. The concentrated flavor precursor mix wassterile filtered and adjusted to pH 5.5-6.0 using NaOH, and used at a 1×concentration in burgers.

TABLE 5 Chemical composition of the flavor precursor mix. Chemicalentity mM Alanine 5.6 Arginine 0.6 Asparagine 0.8 Aspartate 0.8 Cysteine0.8 Glutamic acid 3.4 Glutamine 0.7 Glycine 1.3 Histidine 0.6 Isoleucine0.8 Leucine 0.8 Lysine 0.7 Methionine 0.7 Phenylalanine 0.6 Proline 0.9Threonine 0.8 Tryptophan 0.5 Tyrosine 0.6 Valine 0.9 glucose 5.6 Ribose6.7 Maltodextrin 5.0 Thiamine 0.5 GMP 0.2 IMP 0.6 Lactic acid 1.0creatine 1.0 NaCl 10 KCl 10 Kphos pH 6.0 10

Example 28—Freeze-Alignment to Produce Texturized Protein for Use asMuscle Replicas

This example describes a non-extrusion based method to producetexturized protein materials that can be used in meat replicas.

A muscle tissue-replica was prepared by first preparing a gel of lentilproteins by mixing a 4.5% (w/v) solution of lentil proteins in 20 mMpotassium phosphate buffer pH 7.4+100 mM sodium chloride with 20% (v/v)canola oil (from Jedwards International). The mixture was gelled byheating at 95° C. for 15 minutes and slow cooled to room temperature (atthe rate of 1° C./minute). The gel was then poured into a vessel andfrozen at −40° C. by placing above a liquid nitrogen bath untilcompletely frozen. The frozen material was then dried in a freeze-dryer.When the material was completely dried, the material was stabilized byautoclaving (121° C., 15 minutes). The resulting material is atexturized muscle tissue replica formed with plant proteins.

The aligned muscle replica then was presoaked in water for 5 minutes,cut into pieces of length 3-4 mm and then combined with 10 gadipose-replica, 10 g connective tissue replica and 5 g cold-set gels toform 50 g beef patty replicas. An in-house sensory panel attributedinclusion of freeze-aligned tissue to impart improved fibrous texture tothe patty.

A muscle-tissue replica also was prepared by first formingfreeze-aligned material as described above. After the tissue replica wassteam cooked at 121° C. for 10 minutes, the material was allowed to soakin a solution of heat-denatured pea vicilins (at 6% w/v in 20 mMpotassium phosphate buffer pH 7.4+100 mM sodium chloride, heat denaturedby heating at 95° C. for 30 minutes), 1% equine myoglobin (w/v) (Sigma),and 40% (v/v) canola oil (from Jedwards International). Gelation of themedium was induced by addition of calcium chloride at 20 mM. The samplewas allowed to sit for 5 minutes at room temperature to allow for gelformation. The resulting muscle-replica contained aligned material in acold-set gel reminiscent of beef muscle in steak.

Example 29—Cold Gelation of Proteins for Meat Applications

In one example, a cold-set gel comprising myoglobin was prepared byfirst heat denaturing a 6% (w/v) solution of pea-vicilins in 20 mMpotassium phosphate buffer, pH 7.4 with 100 mM sodium chloride at 100°C. for 30 minutes. The solution was allowed to cool back to roomtemperature. Canola oil (from Jedwards International) and equinemyoglobin (Sigma) were added to a final concentration of 20% (v/v) and1% (w/v), respectively. Gel formation was induced by adding calciumchloride at 20 mM. A 50 g beef patty replica was formed by combining 5 gof the cold gel with 10 g of adipose tissue-replica, 10 g of connectivetissue-replica, and 25 g of muscle tissue-replica. Five mls of a 7%(w/w) solution of crude lentil protein was added to the mixture andpatties were formed.

Example 30—Binding Materials in Meat Replicas

In one example, a beef replica was made by first preparing a coacervatefrom a 3% (w/v) solution of pea vicilins and legumins (vicilin:leguminratio of 3:1) in 20 mM potassium phosphate pH 7.4+100 mM sodiumchloride. Melted palm oil (from Jedwards International) was added to thesolution to a final concentration of 5% and mixed by vortexing. Theemulsion then was acidified by adding hydrochloric acid while stirringto a pH of 5. The slurry was then centrifuged at 5000×g for 10 minutesand the liquid top layer was decanted from the coacervate.

A 50 g beef patty replica was formed by combining the coacervate at 10%with adipose tissue-replica (20%), connective tissue-replica (20%) andmuscle tissue-replica (50%). Five mls of a 7% solution of crude lentilprotein was added to the mixture and patties were formed. Patties thatincluded coacervate as binding material were observed to be morecohesive than patties that did not.

Example 31—Assembly of a Sticky and Noodle Type Ground Tissue Replicaand Burger Replica

Ground tissue replica and burger replicas were prepared using theingredients in Table 6. During all pre-processing steps, the temperatureof the materials was maintained cold (4-15° C.).

TABLE 6 Composition of sticky and noodle type formulation Ingredient %Adipose 26 Soft connective 20.6 Sticky 12 Raw 10 Noodles 10 Hardconnective 10 Flavor and heme solution 10 k-carrageenan 1.4 Total 100

Adipose tissue replica from Example 6 was chilled following the finalheat-cool step into a solid block. Optionally 0.2% by weight of hemeprotein can be added as a 20 mg/ml liquid solution and manually workedinto the adipose. The adipose was then crumbled manually into small bits3-7 mm in diameter while cold.

Soft connective tissue replica from Example 23 was produced by anextrusion process as long string-like pieces. The soft connective waschopped with a mini chopper (Mini-Prep® Plus Processor model DLC-2LCuisinart, Stamford, Conn.) in a single step process. Approximate 200 gof soft connective was placed in the mini chopper and processed on thechop setting for 60 seconds to yield pieces of 1-3 mm in length withragged edges.

Sticky tissue replica and noodles tissue replica (see Examples 25 and26) were produced by an extrusion process as long noodle-like pieces oramorphous pieces, respectively. Raw tissue replica of Example 21 wasprovided from the enzymatic crosslinking process as a solid block. Allthree of these tissue replicas were manually broken down into pieces 1-3cm in diameter.

Hard connective tissue replica from Example 22 was produced from anextrusion process as long string-like pieces. The hard connective tissuereplica was chopped to three levels, named coarse, intermediate, andfine, in a mini chopper (Mini-Prep® Plus Processor model DLC-2LCuisinart, Stamford, Conn.). 160-200 g of hard connective tissue replicawas placed in the mini chopper and processed on the chop setting for 90seconds. One third of the material was removed as the coarse choppedfraction. The material remaining in the chopper was then processed foran additional 60 seconds and one third of the original weight wasremoved as the intermediate chopped fraction. The material remaining inthe chopper was then processed for an additional 30 seconds to producethe fine fraction.

Leghemoglobin was freeze-dried and then reconstituted in a 17× flavorprecursor mix (see Example 27) adjusted to pH 6.0 with 10 N NaOH to makethe flavor and heme protein solution.

Following pre-processing described above, the soft connective, sticky,raw, noodles, hard connective, and 2/3 of the adipose were mixed by handin a bowl. A typical batch size was 100 g to 2000 g. The flavor and hemesolution was then dribbled onto the mixed tissue replicas and mixedgently by hand, then k-carrageenan powder was tsprinkled over themixture and mixed in by hand. During the assembly, grinding, and formingall materials were kept cold (4-15° C.). The mixture was ground using astand mixer fitted with a food grinder attachment (KitchenAid®Professional 600 Series 6 Quart Bowl-Lift Stand Mixer model KP26M1XERand KitchenAid® Food Grinder model FGA, St. Joseph, Mich.) on speedsetting 1. The food grinder material was fed by a screw conveyor past arotating knife installed in front of a fixed hole plate.

The ground tissue replica mix was collected in a bowl and the remaining⅓ of the crumbled adipose was then added to the ground tissue replicamix and mixed in by hand. Approximately 30 g or 90 g portions of groundtissue replica were then formed by hand into round patty shapes. Typicaldimensions for 30 g patties were 50 mm×12 mm. Typical dimensions for 90g patties were 70 mm×18 mm. Patties were refrigerated until cooked.Cooked patties had appearance, texture, and flavor similar to groundbeef as judged by a trained sensory panel. In addition to cooking inpatty format, the ground tissue replica can also be used in a variety ofdishes such as taco filling, casseroles, sauces, toppings, soups, stews,or loaves.

Example 32—Assembly of a Wheat Gluten Containing Ground Tissue Replicaand Burger Replicas

Ground tissue replica and burgers were prepared using the ingredients inTable 7.

TABLE 7 Composition of Wheat Gluten Type formulation Ingredient %Adipose 25 Soft connective 30 Raw tissue replica with heme and flavor 35Wheat gluten 5 Hard connective 5 Total 100

Adipose, soft connective, and hard connective were preprocessed asdescribed in Example 31. During all pre-processing steps, thetemperature of the materials was maintained cold (4-15° C.).

Raw muscle with heme protein and flavor was then prepared as follows.Freeze-dried pea vicilin and pea legumin were dissolved in water and 16×flavor stock. The freeze-dried heme protein was dissolved in thismixture and the pH was adjusted with citric acid to 5.8. A drytransglutaminase preparation (ACTIVA® TI Ajinomoto Fort Lee, N.J.) wasthen added and mixed for about 5 min to fully dissolve. The mixture wasthen allowed to stir for an additional 10 minutes until some increase inviscosity was observed. The soft connective and hard connective werethen added and the mixture was allowed to sit for 1 hour at roomtemperature to cure and form a solid mass.

Wheat gluten powder (vital wheat gluten, Great Northern, item 131100,Giusto's Vita-Grain, South San Francisco, Calif.) then was added to thegelled raw muscle and mixed to distribute. This mixture was thenimmediately ground using a stand mixer fitted with a food grinderattachment as described in the previous example. Ground tissue replicawas then chilled for 5 min at −20° C. Finally, the chopped adipose,pre-chilled to 4° C., was added to the chilled ground tissue replica.

Ground tissue replica mix with adipose tissue replica added was thenformed by hand into two 90 g round patties. 90 g patties are typically70 mm×18 mm. A typical batch size was 180-200 g and produced twopatties. The patties were then allowed to rest at room temperature for30 minutes. After resting the patties can be cooked or refrigerateduntil ready to cook.

Example 33: Generation of Beef Flavor in Replica Burgers by the Additionof Heme and Flavor Precursors

Characteristic flavor and fragrance components in meat are mostlyproduced during the cooking process by chemical reactions molecules(precursors) including amino acids, fats and sugars that are found inplants as well as meat. Flavor precursors along with 1% Leghemoglobinwere added to the muscle component of the burger replicas as indicatedin Table 8. Three replicas, one with no precursors, and two differentmixtures of precursors, along with 80:20 beef were cooked then served toa trained sensory panel to describe the flavor attributes shown in Table9. The addition of precursors increased the beefy flavor, the bloodynotes, overall flavor quantity, and decreased the off notes in thereplica. The replicas and beef sample also were analyzed by GCMS byadding 3 grams of uncooked replica or beef into a GCMS vial. All sampleswere cooked at 150° C. for 3 mins, cooled to 50° C. to extract for 12minutes using GCMS (SPME fiber sampling of headspace). A searchalgorithm analyzed the retention time and mass fingerprint informationto assign chemical names to peaks. In the replica burger with 1% Leghemeoglobin, and precursor mixture 2, 136 beef compounds were created.In Table 10, all the compounds created in the replica burger that werealso identified by GCMS in the beef samples are indicated.

TABLE 8 Flavor precursors added to the beef replicas before cooking.Samples 767 804 929 Precursor No Precursor Additive (mm) Mix 1Precursors Mix 2 Alanine 5.61 5.61 Cysteine 0.83 0.83 Glutamic acid 3.403.40 Leucine 0.76 0.76 Lysine 0.68 0.68 Methionine 0.67 0.67 Tryptophan0.49 0.49 Tyrosine 0.55 0.55 Valine 0.85 0.85 Glucose 5.55 5.55 Ribose6.66 6.66 Lactic acid 1.00 1.00 creatine 1.00 1.00 Thiamine 0.50 0.50IMP + GMP 0.40 0.40 Sucrose 2.00 Fructose 2.00 Xylose 2.00 Maltodextrin0.50% 0.50%

TABLE 9 The sensory score determined by the sensory panel for thereplica burgers and 80:20 beef sample. Sample # Beef 767 804 929 FlavorQuality mean 7.0 3.8 3.2 4.3 STDEV 0.0 1.3 1.0 1.2 Flavor Intensity mean4.3 4.2 4.3 4.3 STDEV 0.8 1.2 1.4 1.2 Flavor: Beefy mean 5.8 3.3 2.3 4.2STDEV 1.0 1.2 0.8 1.2 Flavor: bloody/Metallic mean 4.5 2.0 2.2 3.2 STDEV1.2 1.0 0.9 1.4 Flavor: Savory mean 3.3 3.8 3.7 4.2 STDEV 1.2 1.2 1.21.8 Off Flavors: mean 1.5 2.3 3.5 2.7 chemical/oxidized/beany STDEV 0.81.2 1.8 1.0

TABLE 10 Beef flavor compounds created in replica burger with 1% LegHand precursor mix 2 as detected by GCMS. 3-octen-2-one octanoic acid(Z)-2-decenal, 1-penten-3-ol octane carbon disulfide n-caproic acidvinyl ester octanal butyrolactone 2-acetylthiazole nonanal butanoic acidthiophene 4,7-dimethyl-undecane, 3-methyl-butanal, methyl-thiirane,methyl ethanoate 2-methyl-butanal, thiazole methional butanal styrenemethacrolein 3,6,6-trimethyl-bicyclo[3.1.1]hept-2-ene, isovaleric acidbenzyl alcohol pyrrole isopropyl alcohol 1,3-dimethyl-benzene pyridinehexanoic acid benzene trimethyl-pyrazine 2,2,4,6,6-pentamethyl-heptane,benzaldehyde tetramethyl-pyrazine, 2-methyl-heptane, acetophenonemethyl-pyrazine, heptane acetonitrile ethyl-pyrazine heptanal acetone3-ethyl-2,5-dimethyl-pyrazine furfural acetoin 2,5-dimethyl-pyrazinefuraneol acetic acid ethenyl ester 2,3-dimethyl-pyrazine 3-methyl-furanacetic acid 2-ethyl-5-methyl-pyrazine 2-propyl-furan acetamide2-ethenyl-6-methyl-pyrazine, 2-pentyl-furan acetaldehyde pyrazine2-methyl-furan 4-methyl-5-thiazoleethanol 2-methyl-propanal,2-ethyl-furan 6-methyl-5-hepten-2-one propanal furantrans-2-(2-pentenyl)furan phenylacetaldehyde formamide (E)-4-octene,phenol ethyl acetate 4-cyclopentene-1,3-dione pentanoic acid1-(2-furanyl)-ethanone 4-cyanocyclohexene 3-ethyl-2,2-dimethyl-pentane1-(1H-pyrrol-2-yl)-ethanone dihydro-2-methyl-3(2H)-furanone, pentanaldimethyl trisulfide (E,E)-3,5-octadien-2-one p-cresol dimethyl sulfide3,5-octadien-2-one oxalic acid, butyl propyl ester d-limonene2,2-dimethyl-undecane 1-heptene 1-octen-3-ol toluene1-ethyl-5-methylcyclopentene 1-octanol 1-pentanol 1-butanol 1-hexanol1-octen-3-one 1H-pyrrole-2-carboxaldehyde 2-methyl-1H-pyrrole 2-butanone2-nonanone 3-methyl-2-butenal 2-thiophenecarboxaldehyde2-n-butylacrolein 3-ethylcyclopentanone 2-pyrrolidinone2-methyl-2-heptene 2(5H)-furanone 2-propenal (E)-2-hexenal,dihydro-5-pentyl-2(3H)-furanone 1-hydroxy-2-propanone (E)-2-heptenal,5-ethyldihydro-2(3H)-furanone 1-(acetyloxy)-2-propanone6-methyl-2-heptanone 5-acetyldihydro-2(3H)-furanone 2-pentanone2-heptanone 2,6-dimethylpyrazine (E)-2-octenal 2-furanmethanol(E,E)-2,4-nonadienal 2-octanone 3-ethyl-2-1,4-dioxin(E,E)-2,4-heptadienal (E)-2-nonenal 3-ethyl-2-methyl-1,3-hexadiene(E,E)-2,4-decadienal 8-methyl-1-undecene 2-butenal2,3-dimethyl-5-ethylpyrazine 1-propanol 1-penten-3-one

Example 34—Removal of Off-Flavors from Plant Protein Solutions

i) Synthesis of Ligand Modified LOX Removal Resin

A 1 mL settled volume of CM Sepharose resin (Sigma Aldrich Catalog#CCF100) was loaded into a BioRad minicolumn. 3 mL of 50 mM MES(2-morpholinoethanesulfonic acid) buffer preset to a pH range of 5.5 to6 was allowed to pass through the resin bed. Separately, to 1 mL of thesame buffer was added in succession, 0.044 mL of4,7,10-trioxa-1,13-tridecanediamine, 0.030 mL of 12 N HCl, 23 mg of NHS(N-hydroxy-succinimide), and 38 mg of EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) withdissolution after each addition. The resulting solution was added to thetop of the column bed and allowed to gravity flow through and theeffluent was collected. The resulting effluent was returned to the topof the column bed. The cycle of addition of solution, elution and returnwas done four times. When the last elution was finished, 3 mL of 50 mMIVIES buffer preset to a pH of 5.5 to 6 was allowed to gravity flowthrough the column. Linoleic acid (0.03 mL) was dissolved in 0.5 mL DMF(N,N-dimethylformamide), followed in sequence by 12 mg NHS, 19 mg EDC,and 0.5 mL of 50 mM MES buffer preset to a pH of 5.5 to 6. The NHS/EDCmixture was shaken to mix, resulting in a two-phase liquid which wasapplied to the top of the column and eluted through the resin bed,allowed to gravity flow through the column and effluent collected. Theeffluent was returned to the top of the column bed. The cycle ofaddition of solution, elution and return was done four times. Once thelast collection was finished a solution of 70% ethanol in water (5 mL)was added to the top of the column, followed by 3 mL of 0.1 M sodiumhydroxide. This was followed by 0.1 M buffer of potassium phosphatepreadjusted to pH 7 to 8.

ii) Removal of Off-Flavors from Pea Proteins Using the Ligand ModifiedLOX Removal Resin.

A solution of pea proteins (30 mls at 20 mg/ml protein concentration in20 mM potassium phosphate buffer pH 7.4, 100 mM sodium chloride) waspassed through a 100 ml resin bed described above. All unbound materialwas collected and the resin further washed with 200 mls of buffer. Bothfractions were combined and resin was stripped of bound protein bywashing with 2 bed volumes of 20 mM potassium phosphate, 1M sodiumchloride. Resin bed was regenerated by washing with 2 bed volumes of0.1M NaOH followed by 3 bed volumes of water and re-equilibration withbuffer.

Depletion of LOX activity in the pooled unbound fraction was confirmedby assaying for enzyme activity. Sodium linoleate was used as substratefor LOX and formation of hydroperoxide intermediates was monitored byabsorbance at 234 nm. Assays were performed at pH9 in 50 mM sodiumborate buffer. Assays confirmed depletion of LOX activity in the pooledunbound fraction. LOX was removed from resin in washes with 0.5M and 1Msodium chloride.

Improvement in flavor in the LOX depleted protein solutions (both as-isand incubated with canola oil at 10%) was confirmed by a panel of 4tasters. Samples of pea proteins at same concentration, but not depletedof LOX was used at control. Tasters described LOX -depleted samples asmild tasting and control samples as having beany, planty taste. Inaddition, GC-MS analysis of samples showed 5× reduction in overallvolatiles in the LOX-depleted samples.

Reduction of Off-Flavors Using Dialysis and Activated Carbon

A solution of pea albumins (30 mls at 40 mg/ml in 20 mM potassiumphosphate buffer pH 7.4, 100 mM sodium chloride) was dialyzed against100 volumes of buffer overnight at 4° C. The solution was then pouredover a bed of activated carbon (100-mesh, Sigma-Aldrich) prewetted withbuffer. The slurry was centrifuged at 5000×g for 10 minutes and thesupernatant containing protein was decanted off. This solution wastested for improvement in flavor by both taste and GC-MS. Improvedflavor (less bitter, less soapy flavor) was noted by tasters and GC-MSconfirmed a >2× reduction in volatile compounds; in particular, samplestreated with activated carbon showed a decrease in C6- and C7 compounds(e.g., 1-heptanal, 2-heptenal, or 2-heptanone) that are associated withgreen/grassy/planty flavors.

Reduction of Off-Flavors Using Antioxidants and/or LOX Inhibitors

A 7% solution of pea-vicilins was heated with coconut oil (20%) to 95°C. for 15 min in the presence of antioxidants or LOX inhibitors andcompared for off-flavors sample against a control without anyantioxidants or LOX inhibitors. In a similar experiment, soymilk washeated in the presence of antioxidants or LOX inhibitors and tastedin-house for off-flavors. Table 11 summarizes off-flavors noted bytasters. Both epigallocetechin gallate and propyl gallate were effectiveat minimizing off-flavors in pea samples. However, in case of soymilk,epigallocatechin gallate did not appear to reduce beaniness; propylgallate and α-tocopherol were found to slightly improve flavor insoymilk.

TABLE 11 Compound soymilk Pea α-tocopherol Slightly improved flavorOxidized oil caffeic acid (0.02%) Beany Oxidized oil epigallocatechingallate Beany Improved flavor propyl gallate (0.02%) Slightly improvedflavor Improved flavor β-carotene Beany Oxidized oil

Example 35—Adipose Replica with Lecithin Gradient

Lecithin (SOLEC™ Deoiled Soy Lecithin, The Solae Company, St. Louis,Mo.) was prepared at a concentration of 50 mg/mL in 20 mM potassiumphosphate, 100 mM NaCl, pH 8.0 buffer and sonicated (Sonifier AnalogCell Disruptor model 102C, BRANSON Ultrasonics Corporation, Danbury,Conn.) for 30 seconds. Moong protein was supplied as a liquid in 20 mMpotassium phosphate, 100 mM NaCl, pH 8.0 buffer. Coconut oil was meltedby heating to 50-70° C. and held warm until needed. The coconut oil,buffered protein solution, additional buffer, and lecithin slurry weremixed at 70° C., and an emulsion was formed using a hand heldhomogenizer. The emulsion was then heat set by placing the tube into a95° C. waterbath for a total of 5 mins. The tube was then removed fromthe waterbath and stored at 4° C. for twelve hours or longer prior toanalysis.

To observe the effect of lecithin on the adipose tissue replica replica,a formulation was prepared that contained 1% w/v moong protein and 75%v/v coconut oil, with the lecithin amount increasing from 0%, 0.05%,0.25%, 0.5%, and 1.0% w/v.

Properties of the adipose replica were measured by weighing smallportions of the material and forming uniform round balls, which werethen cooked on a non-stick pan with temperature slowly ramping up to150° C. The temperature of the pan at which fat visibly released fromthe balls was measured as the fat release temperature. After cooking tocompletion, the point at which fat no longer released, the total fatreleased was measured.

An increase in the amount of lecithin in the adipose replica correlatedwith an increase in the percent fat released, and with a decrease in thefat release temperature (see FIGS. 2A and 2B). With 0% lecithin, therewas an average of 40% fat released, and when lecithin was increased to0.05%, there was an average of 82% fat released, which further increasedto 88% with 0.25% lecithin, then leveled off to an average of 60% withfurther increases in lecithin. With 0% lecithin, a high temperature of217° C. was required to begin the fat release. The fat releasetemperature decreased to 122° C. with 0.25% lecithin, then leveled offto an average of 62° C. with further increases of lecithin.

Firmness of the adipose replica was measured by texture analyzer (TA XTplus). A probe penetrated a flat surface of the adipose replica, and theforce at 2 mm penetration was recorded. A small amount of lecithin(0.05%) increased the firmness, and at 0.25% and above, the firmness ofthe adipose replica decreased. See FIG. 2C.

Example 36—Adipose Replica with Varying Vegetable Oil Type

To observe the effect of vegetable oil type on the adipose replica, aformulation was prepared that contained a 1.5% w/v moong protein and0.05% w/v lecithin, and 75% v/v of different vegetable oils (canola oil,cocoa butter, coconut oil, and olive oil) using the same methodology asExample 35. Properties of the adipose replica were measured by weighingsmall portions of the material and forming uniform round balls, whichwere then cooked on a non-stick pan with temperature slowly ramping upto 250° C. The temperature of the pan at which fat visibly released fromthe balls was measured as the fat release temperature. After cooking tocompletion, the point at which fat no longer released, the total fatreleased was measured.

Varying the vegetable oil type had a large effect on the adiposereplica. Oils with a higher amount of unsaturated fats, including canolaand rice bran oils, released very little fat (1 and 2% fat released, seeFIG. 3), while oils with a higher amount of saturated fats, includingcocoa butter and coconut oil released significantly more fat (30% and50% fat released, see FIG. 4). The adipose replicas with canola and ricebran oils required a temperature greater than 250° C. to melt (notmeasured), while the adipose replicas with cocoa butter and coconut oilreleased fat at a lower temperature (82° C. and 137° C.).

Example 37—Adipose Replica Made with Coacervate

Lecithin (SOLEC™ F Deoiled Soy Lecithin, The Solae Company, St. Louis,Mo.) was prepared at a concentration of 50 mg/ml in 20 mM potassiumphosphate, 100 mM NaCl, pH 8.0 buffer and sonicated (Sonifier AnalogCell Disruptor model 102C, BRANSON Ultrasonics Corporation, Danbury,Conn.) for 30 seconds. Pea legumin and pea vicilin proteins in 20 mMpotassium phosphate, 100 mM NaCl, pH 8.0 buffer, were mixed to a 1:1ratio. Cocoa butter was melted by heating to 70° C. and held warm untilneeded. The cocoa butter was added to 2% and 10% w/v to the proteinmixtures, and was added at 60° C. to maintain cocoa butter in the liquidstate. While the solutions were still warm, the mixtures were sonicated1-3 minutes, until cocoa butter particles were visibly emulsified. ThepH of the samples was adjusted to 5.5 with HCl, and the mixtures turnedinto a milky white color, then centrifuged at 5,000×g for ten minutes.After centrifugation, a pellet was collected, comprising a coacervate ofprotein and cocoa butter. The 2% fat coacervates were sticky andstretchy, while the 10% fat coacervates were fatty and pliable. Thecoacervates were sealed in plastic and subjected to high pressureprocessing (HPP). The sample was sealed in a heat-sealable food-saverplastic bag and then subject to high pressure processing (85 k psi for 5minutes in an Avure 2L Isostatic Food Press). After HPP, the 2%coacervate samples formed a semi-firm, cohesive material. The 10%coacervate samples were crumbly, soft, and oily.

Properties of the processed coacervate samples were measured by breakingoff small portions of the material and cooking on a non-stick pan withtemperature slowly ramping up to 250° C. The coacervate samples did notrelease any fat upon cooking to this temperature.

Other Embodiments

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. (canceled)
 2. A method for imparting a color change to a consumableproduct, the method comprising: including about 0.01% to about 5% byweight of a plant heme-containing protein, an algal heme-containingprotein, a fungal heme-containing protein, a protozoan heme-containingprotein, or a bacterial heme-containing protein into the consumableproduct, wherein the consumable product transitions from a substantiallypink or red color in an uncooked state to a substantially brown colorduring cooking.
 3. The method of claim 2, wherein the consumable productcomprises one or more of an emulsifier, a gelling agent, a fiber, ashelf life extender, an anti-oxidant, a starch, a gum, and combinationsthereof.
 4. The method of claim 3, wherein the shelf life extender isselected from one or more of a nitrite, sodium metabisulfite, BOMBAL®,vitamin E, rosemary extract, green tea extract, and a catechin.
 5. Themethod of claim 3, wherein the anti-oxidant is selected from one or moreof glutathione, vitamin C, vitamin A, vitamin E, caffeic acid,epigallocatechin gallate, propyl gallate, and beta-carotene.
 6. Themethod of claim 3, wherein the gum is selected from one or more ofxanthan gum and carrageenan.
 7. The method of claim 2, wherein theconsumable product comprises a sugar selected from the group consistingof glucose, ribose, sucrose, fructose, xylose, maltodextrin, andcombinations thereof, and a sulfur compound selected from methionine,cysteine, thiamine, and combinations thereof.
 8. The method of claim 2,wherein the consumable product comprises about 0.1% to about 1% byweight of the heme-containing protein.
 9. The method of claim 2, whereinthe plant heme-containing protein is from a plant selected from thegroup consisting of Nicotiana tabacum, Nicotiana sylvestris, Zea mays,Arabidopsis thaliana, Glycine max, Cicer arietinum, Pisum sativum,Phaseolus vulgaris, Vigna unguiculata, Vigna radiata, Lupinus albus,Medicago sativa, Brassica napus, Triticum sps., Gossypium hirsutum,Oryza sativa, Zizania sps., Helianthus annuus, Beta vulgaris, Pennisetumglaucum, Chenopodium sp., Sesamum sp., Linum usitatissimum, and Hordeumvulgare.
 10. The method of claim 2, wherein the algal heme-containingprotein is from Chlamydomonas eugametos.
 11. The method of claim 2,wherein the fungal heme-containing protein is from a fungus selectedfrom the group consisting of Saccharomyces cerevisiae, Pichia pastoris,Magnaporthe oryzae, Fusarium graminearum, and Fusarium oxysporum. 12.The method of claim 2, wherein the protozoan heme-containing protein isfrom a protozoa selected from the group consisting of Parameciumcaudatum and Tetrahymena pyriformis.
 13. The method of claim 2, whereinthe protozoan heme-containing protein is from a ciliate.
 14. The methodof claim 2, wherein the bacterial heme-containing protein is from abacteria selected from the group consisting of Escherichia coli,Bacillus subtilis, Bacillus megaterium, Synechocistis sp., Aquifexaeolicus, Methylacidiphilum infernorum, and Thermophilus spp.
 15. Themethod of claim 2, wherein the consumable product further comprises oneor more plant proteins.
 16. The method of claim 15, wherein the one ormore plant proteins comprise an extruded protein.
 17. The method ofclaim 15, wherein the one or more plant proteins comprise legumeproteins.
 18. The method of claim 17, wherein the legume proteins areselected from pea proteins and lentil proteins.
 19. The method of claim18, wherein the pea proteins comprise pea albumin proteins.
 20. Themethod of claim 15, wherein the one or more plant proteins compriseRubisco.
 21. The method of claim 15, wherein the one or more plantproteins are present in the consumable product in an amount of between1% and 30% by weight of the consumable product.